Conjugates of soluble peptidic compounds with membrane-binding agents

ABSTRACT

The present invention provides, among other things, soluble derivatives of soluble polypeptides that incorporate membrane binding elements. Methods of making these soluble derivatives, and methods of using these soluble derivatives also are provided.

This application is a continuation-in-part of U.S. Ser. No. 09/214,913,filed Mar. 16, 1999, which is a 371 of PCT/EP97/03715, filed Jul. 8,1997. The entirety of these applications is hereby incorporated byreference.

This invention relates to polypeptide derivatives, their use in therapyand methods and intermediates for their production.

Essentially all protein drugs are administered as solutions and functionin vivo in the solution phase. In biochemist and pharmacology, however,a large number of control and mediator proteins are associated with orfunction within or on the plasma membranes of cells. Except for soluble,truncated versions of one class of these molecules. nomembrane-associated proteins have been developed as therapeutic agents.There are two main reasons for this situation. Firstly. overexpressionof proteins that are retained in the membranes of the producer cells islimited by the the low capacity of membranes for proteins and often bythe toxic effects of retention when expression is intrinsicallyefficient. Secondly, extraction of these proteins from membranesrequires detergents or organic solvents, often results in inactivationof the protein. leads to difficulties in achieving the high purityneeded for drug use and usually gives a product which is hard toformulate for intravenous administration. In addition. retention of veryhydrophobic membrane anchoring elements may cause proteins to associatestrongly with lipid-binding proteins in blood when administredintravenously thus preventing access to cell membranes.

Soluble, (truncated versions of membrane-associated proteins overcomethe production difficulties associated with full length proteins.However such truncated molecules lack the membrane binding capabilityand specificity of the full length proteins which properties may beadvantageous or even essential to the desired therapeutic activity.

The main classes of interaction of proteins with membranes can besummarised as follows:

1. Direct and specific interactions with phospholipid bead groups orwith other hydrophilic regions of complex iipids or indirectly withproteins already inserted in the membrane. The latter may include allthe types of intrinsic membrane protein noted below and suchinteractions are usually with exvacellular domains or Sequence loops ofthe membrane proteins;

2. Through anchoring by a single hydrophobic tranimembrane helicalregion near the terminus of the protein. These regions commonly prescnta hydrophobic face around the entire circumference of the helix cylinderand transfer of this structure to the hydrophilic environment of bulkwater is energetically unfavourable.

3. Further anchoring is often pmvided by a shon sequence of generallycationic aminoacids at the cytoplasmic side of the membrane, C-terminalto the transmembrane helix;

4. Through the use of multiple (normally 2-12 and commonly 4,7 and 10)transmembrane regions which are usually predicted to be helical ornear-helical. Although these regions are normally hydrophobic overall,they frequently show some amphipathic behaviour—an outer hydrophobicface and an inner more hydrophilic one being identifiable within a helixbundle located in the lipid bilayer;

5. Through postranslationally linked phosphatidyl inositol moeities(GPI-anchors). These are generated by a specific biosynthetic pathwaywhich recognises and removes a specific stretch of C-terminal aminoacidsand creates a membrane-associating diacyl glycerol unit linked via ahydrophilic carbohydrate spacer to the polypeptide;

6. In a related process, single fatty acid groups such as myristoyl,palmitoyl or prenyl may be attached postranslationally to one or moresites in a protein (usually at N- or C-termini). Again, amino acids(such as the C-terminal CAAX box in Ras proteins) may be removed.

Artificial membranes are considered to be lipid complexes that mimic thebasic properties of the cell membrane, i.e., a lipid vacuole with anaqueous interior and a surface chemistry that resembles the cellmembrane. The artificial membrane typically contains phospholipids ormimics thereof and may be unilemellar or bilemellar and the outersurface will contain charged groups similar to the choline groups of themost abundant phospholipid. The prototype artificial membrane is knownas a liposome and the technologies for the construction of liposomesincluding the incorporation of therapeutically useful agents into themis well known to those in the art. Liposomes have been evaluated in anumber of disease states and liposomes containing the anti-fungalAmphotericin are commercially available. In addition, proteoliposomeshave been described. For example, the use of immunoliposomesencapsulating amphotericin B has been reported to be of benefit in thetreatment of experimental fungal infections in animal models (e.g.Hospenthal, D. et al (1989) J. Med. Microbiol. 30 193-197; Dromer, F. etal (1990) Antimicrob. Agents Chemother. 34 2055-2060).

Mimics of natural or artificial membranes are often related in structureand will mimic one or more properties of the membrane. One such exampleis the provision of an artificial surface having pendant groups whichmimic the phospholipid zwitterionic groups which are found on theoutside of cell surfaces. For example WO92/06719 (BiocompatiblesLimited) discloses natural and synthetic phospholipids which may becoated on an artificial surface, e.g. a device which. in use, will comeinto contact with protein-containing or biological fluids, to provideimproved biocompatibility and haemocompatibility and WO 94/16749discloses additional zwitterionic groups that may be used to improvebiocompatibility in a similar way.

The present invention provides a soluble derivative of a solublepolypeptide, said derivative comprising two or more heterologousmembrane binding elements with low membrane affinity covalentlyassociated with the polypeptide which elements are capable ofinteracting, independently and with thermodynamic additivity, withcomponents of cellular or artificial membranes exposed to extracellularfluids.

By ‘heterologous’ is meant that the elements are not found in the nativefull length protein from which a soluble protein may be derived.

By ‘soluble polypeptide’ is meant a truncated derivative of a fulllength protein which lacks its natural membrane binding capability,and/or a polypeptide which has a solubility level in aqueous mediaof >100 μg/ml.

By ‘membrane binding element with low membrane affinity’ is meant thatthe element has only moderate affinity for membranes, that is adissociation constant greater than 0.1 μM, preferably 1 μRM-1 mM. Theelements preferably have a size <5 kDa.

The derivative should incorporate sufficient elements with lowaffinities for membrane components to result in a derivative with a high(preferably 0.01-10 nM dissociation constant) affinity for specificmembranes. The elements combine so as to create an overall high affinityfor the particular target membrane but the combination lacks such highaffinity for other proteins for which single elements may be(low-affinity) ligands.

The elements should be chosen so as to retain useful solubility inpharmaceutial formulation media, preferably >100 μg/ml. Preferably atleast one element is hydrophilic.

The invention thus promotes localisation of a therapeutic protein atcellular membranes and thereby provides one or more of severalbiologically significant effects with potential therapeutic advantagesincluding:

Potency: If the protein is a receptor and an agonist or antagonistactivity is localised on the same surface as the receptor itself, anincrease in effective concentration may result from the reduction in thediffusional degrees of freedom.

Pharmacokinetics and dosing frequency: Interaction of a derivatisedprotein with long-lived cell types or serum proteins would be expectedto prolong the plasma residence time of the protein and produce a depoteffect through deposition on cell surfaces.

Specificity: Many clinically important pathological processes areassociated with specific cell types and tissues (for example thevascular endothelium and the recruitment thereto of neutrophils bearingthe sialyl Lexis, antigen to ELAM-1, see below). Hence targeting themodified protein to regions of membrane containing pathology-associatedmembrane markers may improve the therapeutic ratio of the proteintargeted.

The derivatives of the invention may be used in association withartificial membranes or mimics thereof to allow delivery of thetherapeutic protein to sites where it will provide therapeutic benefit.For example, polypeptides associated with liposomes formed by contactingliposomes with a derivative of the invention may be more stable than thefree polypeptide. The liposome may incorporate a therapeutic agent, forexample an antiflammatory or cytotoxic agent. The polypeptide derivativeof the invention may thus be used to target the therapeutic agents Whenthe polypeptide is itself a therapeutic agent. the liposome incorporatedtherapeutic agent may be used to enhance further the efficacy ortolerability of the therapy.

Association of derivatives of the invention with mimics of cellmembranes may be used to concentrate the therapeutic protein at siteswhere therapeutically useful concentrations of underivatised proteinmight be difficult to achieve. For example, indwelling medical devicescoated with mimics of the phospholipid zwitterionic groups which arefound on the outside of cell surfaces, such as those disclosed inWO92/06719 and WO 94/16749, may be additionally treated with derivativesof the invention. For example complement inhibitors derivatised inaccordance with the invention could be incorporated into the outersurface of indwelling catheters or hip replacements or heart valves inorder to minimise development of inflammation associated with theseoperations.

It will be appreciated that all associations of heterologous amino acidsequences with a polypeptide which is a soluble derivative of a humanprotein will need to be assessed for potential immunogenicity,particularly where the amino acid sequence is not derived from a humanprotein. The problem can be minimised by using sequences as close aspossible to known human ones and through computation of secondarystructure and antigenicity indices.

Examples of protein therapeutic agents which may be modified accordingto the invention include but are not restricted to the following:

Therapeutic Base Protein Cell Target Application IL-4 Y124D muteinB-cells Anti-allergy (IL-4 antagonist) Plasminogen activatorsErythrocytes, Prevention of venous e.g. Prourokinase, vascularthrombosis streptokinase, tissue-type endothelium plasminogen activator,reteplase Leptin Choroid plexus, Weight loss (agonist) HypothalamusComplement inhibitors* Vascular Ischaemic injury, endothelium,transplantation, Myocytes, inflammation Erythrocytes, Lymphocytes scFvantibody against Eosinophils Asthma, cytokines (IL-1, IL-, IL-5,allergic disease IL-6) Protein C Vascular Prevention of venousendothelium thrombosis Antibodies against CD4, LymphocytesImmunosuppresion B7/CD28, CD3/TCR, CD11b(CR3) Interferon-β andLymphocytes Immunomodulation, derivatives multiple sclerosis *Complementregulatory proteins e.g.: CR1 (CD35); DAF (CD55); MCP (CD46); CD59;Factor H; and C4 binding protein; and hybrids or muteins thereof such asCR1-CD59 (S.G.El Feki and D.T. Fearon Molecular Immunology 33 (supp 1).p 57, 1996). MCP-DAF (P.J. Higgins et al, J. Immunology. 158, 2872-2881,1997) and soluble CR1 polypeptide fragments. The derivative preferablycomprises two to eight, more preferably two to four membrane bindingelements. Membrane binding elements are preferably # selected from:fatty acid derivatives such as fatty acyl groups; basic amino acidsequences; ligands of known integral membrane proteins; sequencesderived from the complementarity-determining region of monoclonalantibodies raised against epitopes of membrane proteins; membranebinding sequences identified through screening of random chemical orpeptide libraries. The selection of suitable combination of membranebinding elements will be guided by the nature of the target cellmembrane or # components thereof.

Suitable fatty acid derivatives include myristoyl (12 methylene units)which is insufficiently large or hydrophobic to permit high affinitybinding to membranes. Studies with myristoylated peptides (eg R. M.Peitzsch & S. McLaughlin, Biochemistry, 32, 10436-10443, 1993)) haveshown that they have effective dissociation constants with model lipidsystems of −10⁴ M and around 10 of the 12 methylene groups are buried inthe lipid bilayer. Thus, aliphatic acyl groups with about 8 to 18methylene units, preferably 10-14, are suitable membrane bindingelements. Other examples of suitable fatty acid derivatives includelong-chain (8-18, preferably 10-14 methylene) aliphatic amines andthiols, steroid and farnesyl derivatives.

Membrane binding has been found to be associated with limited(single-site) modification with fatty acyl groups when combined with acluster of basic amino acids in the protein sequence which may interactwith acidic phospholipid head groups and provide the additional energyto target membrane binding. This combination of effects has been termedthe ‘myristoyl-electrostatic switch’ (S.McLaughlin and A.Aderem, TIBS,20,272-276, 1994; J. F.Hancock et al, Cell, 63, 133-139, 1990). Thus, afurther example of suitable membrane binding elements are basic aminoacid sequences such as those found in proteins such as Ras and MARCKS(myristoylated alanine-rich C-kinase substrate, P. J. Blackshear, J.Biol. Chem., 268, 1501-1504, 1993) which mediate the electrostatic‘switch’ through reversible phosphorylation of serine residues withinthe sequence and a concomitant neutralisation of the net positivecharge. Such sequences include but are not restricted to consecutivesequences of Lysine and Arginine such as (Lys)n where n is from 3 to 10,preferably 4 to 7. (SEQ ID NO: 53).

Suitable examples of amino acid sequences comprising basic amino acidsinclude:

(i) DGPKKKKKKSPSKSSG (SEQ ID NO: 8)

(ii) GSSKSPSKKKKKKPGD (SEQ ID NO: 9)

(iii) SPSNETPKKKKKRFSFKKSG (SEQ ID NO: 10)

(iv) DGPKKKKKKSPSKSSK (SEQ ID NO: 11)

(v) SKDGKKKKKKSKTK (SEQ ID NO: 12)

(N-terminus on left)

Sequences i) to v) are examples of electrostatic switch sequences.

Examples of amino acid sequences derived from ligands of known integralmembrane proteins include RGD-containing peptides such as GRGDSP (SEQ IDNO: 13) which are ligands for the α_(db)β_(S) integrin of human plateletmembranes. Another example is DGPSEILRGDFSS (SEQ ID NO: 17) derived fromhuman fibrinogen alpha chain, which binds to the GpIIb/IIIa membraneprotein in platelets.

Further examples of such sequences include those known to be involved ininteractions between membrane proteins such as receptors and the majorhistocompatibility complex. An example of such a membrane protein ligandis the sequence GNEQSFRVDLRTLLRYA (SEQ ID NO: 21) which has been shownto bind to the major histocompatibility complex class 1 protein (MHC-1)with moderate affinity (L.Olsson et al, Proc. Natl. Acad. Sci. USA. 91,9086-909, 1994).

Yet further examples of such sequences employ a membrane insertiveaddress specific for T-cells. Such sequence is derived from the knowninteraction of the transmembrane helix of the T-cell antigen receptorwith CD3 (Nature Medicine 3, 84-88, 1997). Examples are peptidescontaining the sequence GFRILLLKV (SEQ ID NO: 22) such as:

SAAPSSGFRILLLKV (SEQ ID NO: 24)

AAPSVIGFRILLLKVAG (SEQ ID NO: 32)

An example of a ligand for an integral membrane protein is thecarbohydrate ligand Sialyl Lewis' which has been identified as a ligandfor the integral membrane protein ELAM-1 (M. L. Phillips et al, Science,250, 1130-1132, 1990 & G. Walz et al, Ibid, 250, 1132-1135,1990).

Sequences derived from the complementarity-determining regions ofmonoclonal antibodies raised against epitopes within membrane proteins(see, for example, J. W. Smith et al, J.Biol.Chem. 270, 30486-30490,1995) are also suitable membrane binding elements. as are bindingsequences from random chemical libraries such as those generated in aphage display format and selected by biopanning operations in vitro (G.F.Smith and J. K.Scott, Methods in Enzymology, 217H, 228-257,1993) or invivo (R.Pasqualini & E.Ruoslahti, Nature, 380, 364-366, 1996).

Optionally, conditional dissociation from the membrane may beincorporated into derivatives of the invention using mechanisms such aspH sensitivity (electrostatic switches), regulation through metal ionbinding (using endogenous Ca²⁺, Zn²⁺ and incorporation of ion bindingsites in membrane binding elements) and protease cleavage (e.gplasminolysis of lysine-rich membrane binding sequences to release andactivate prourokinase)

Preferred derivatives of this invention have the following structure:

[P]—(L—[W)]_(n)—X

in which:

P is the soluble polypeptide,

each L is independently a flexible linker group,

each W is independently a peptidic membrane binding element,

n is an integer of 1 or more and

X is a peptidic or non-peptidic membrane-binding entity which may becovalently linked to any W.

Peptidic membrane binding elements are preferably 8 to 20 amino acidslong and elements W are preferably located sequentially either at the Nor C terminus of the soluble polypeptide. The amino acid sequences arelinked to one another and to the soluble peptide by linker groups whichare preferably selected from hydrophilic and/or flexible aminoacidsequences of 4 to 20 aminoacids; linear hydrophilic synthetic polymers;and chemical bridging groups.

Peptide linkages may be made chemically or biosynthetically byexpression of appropriate coding DNA sequences. Non peptide linkages maybe made chemically or enzymatically by post-translational modification.

The polypeptide portion of the derivatives of the invention may beprepared by expression in suitable hosts of modified genes encoding thesoluble polypeptide of interest plus one or more peptidic membranebinding elements and optional residues such as cysteine to introducelinking groups to facilitate post translational derivatisation withadditional membrane binding elements.

In a further aspect, therefore, the invention provides a process forpreparing a derivative according to the invention which processcomprises expressing DNA encoding the polypeptide portion of saidderivative in a recombinant host cell and recovering the product andthereafter post translationally modifying the polypeptide to chemicallyintroduce membrane binding elements.

In particular, the recombinant aspect of the process may comprise thesteps of:

i) preparing a replicable expression vector capable, in a host cell, ofexpressing a DNA polymer comprising a nucleotide sequence that encodessaid polypeptide portion;

ii) transforming a host cell with said vector;

iii) culturing said transformed host cell under conditions permittingexpression of said DNA polymer to produce said polypeptide; and

iv) recovering said polypeptide.

Where the polypeptide portion is novel, the DNA polymer comprising anucleotide sequence that encodes the polypeptide portion as well as thepolypeptide portion itself and S-derivatives thereof, also form part ofthe invention. In particular the invention provides a polypeptideportion of a derivative of the invention comprising the soluble peptidelinked by a peptide bond to one peptidic membrane binding element and/orincluding a C-terminal cysteine, and DNA polymers encoding thepolypeptide portion.

The recombinant process of the invention may be performed byconventional recombinant techniques such as described in Sambrook etal., Molecular Cloning: A laboratory manual 2nd Edition. Cold SpringHarbor Laboratory Press (1989) and DNA Cloning vols I, II and m (D. M.Glover ed., IRL Press Ltd).

The invention also provides a process for preparing the DNA polymer bythe condensation of appropriate mono-, di- or oligomeric nucleotideunits.

The preparation may be carried out chemically, enzymatically, or by acombination of the two methods, in vitro or in vivo as appropriate.Thus, the DNA polymer may be prepared by the enzymatic ligation ofappropriate DNA fragments, by conventional methods such as thosedescribed by D. M. Roberts et al., in Biochemistry 1985, 24, 5090-5098.

The DNA fragments may be obtained by digestion of DNA containing therequired sequences of nucleotides with appropriate restriction enzymes,by chemical synthesis, by enzymatic polymerisation, or by a combinationof these methods.

Digestion with restriction enzymes may be performed in an appropriatebuffer at a temperature of 20°-70° C., generally in a volume of 50 μl orless with 0.1-10 μg DNA.

Enzymatic polymerisation of DNA may be carried out in vitro using a DNApolymerase such as DNA polymerase 1 (Klenow fragment) in an appropriatebuffer containing the nucleoside triphosphates dATP, dCTh, dGTP and dTTPas required at a temperature of 10°-37° C., generally in a volume of 50μl or less.

Enzymatic ligation of DNA fragments may be carried out using a DNAligase such as T4 DNA ligase in an appropriate buffer at a temperatureof 4° C. to 37° C., generally in a volume of 50 μl or less.

The chemical synthesis of the DNA polymer or fragments may be carriedout by conventional phosphotriester, phosphite or phosphoramiditechemistry, using solid phase techniques such as those described in‘Chemical and Enzymatic Synthesis of Gene Fragments—A Laboratory Manual’(ed. H. G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or inother scientific publications, for example M. J.Gait, H. W. D. MatthesM. Singh, B. S. Sproat and R. C. Titmas, Nucleic Acids Research, 1982,10, 6243; B. S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24,5771; M. D. Matteucci and M. H. Caruthers, Tetrahedron Letters, 1980,21, 719; M. D. Matteucci and M. H. Caruthers, Journal of the AmericanChemical Society, 1981, 103, 3185; S. P. Adams et at., Journal of theAmerican Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biernat, J.McMannus and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.W. D. Matthes et al., EMBO Journal, 1984, 3, 801. Preferably anautomated DNA synthesiser (for example, Applied Biosystems 381ASynthesiser) is employed.

The DNA polymer is preferably prepared by ligating two or more DNAmolecules which together comprise a DNA sequence encoding thepolypeptide.

The DNA molecules may be obtained by the digestion with suitablerestriction enzymes of vectors carrying the required coding sequences.

The precise structure of the DNA molecules and the way in which they areobtained depends upon the structure of the desired product The design ofa suitable strategy for the construction of the DNA molecule coding forthe polypeptide is a routine matter for the skilled worker in the art.

In particular, consideration may be given to the codon usage of theparticular host cell. The codons may be optimised for high levelexpression in E. coli using the principles set out in Devereux et al.,(1984) Nucl. Acid Res., 12, 387.

The expression of the DNA polymer encoding the polypeptide in arecombinant host cell may be carried out by means of a replicableexpression vector capable, in the host cell, of expressing the DNApolymer. Novel expression vectors also form part of the invention.

The replicable expression vector may be prepared in accordance with theinvention, by cleaving a vector compatible with the host cell to providea linear DNA segment having an intact replicon, and combining saidlinear segment with one or more DNA molecules which, together with saidlinear segment, encode the polypeptide, under ligating conditions.

The ligation of the linear segment and more than one DNA molecule may becarried out simultaneously or sequentially as desired.

Thus, the DNA polymer may be preformed or formed during the constructionof the vector, as desired. The choice of vector will be determined inpart by the host cell, which may be prokaryotic, such as E. coli, oreukaryotic, such as mouse C127, mouse myeloma, chinese hamster ovary,fungi e.g. filamentous fungi or unicellular ‘yeast’ or an insect cellsuch as Drosophila. The host cell may also be in a transgenic animal.Suitable vectors include plasmids, bacteriophages, cosmids andrecombinant viruses derived from, for example, baculoviruses orvaccinia.

The DNA polymer may be assembled into vectors designed for isolation ofstable transformed mammalian cell lines expressing the fragment e.g.bovine papillomavirus vectors in mouse C127 cells, or amplified vectorsin chinese hamster ovary cells (DNA Cloning Vol. II D. M. Glover ed. IRLPress 1985; Kaufman, R. J. et at.. Molecular and Cellular Biology 5,1750-1759, 1985; Pavlakis G. N. and Hamer, D. H. Proceedings of theNational Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, D. V. etal., European Patent Application No. 0093619, 1983).

The preparation of the replicable expression vector may be carried outconventionally with appropriate enzymes for restriction, polymerisationand ligation of the DNA, by procedures described in, for example,Sambrook et al., cited above. Polymerisation and ligation may beperformed as described above for the preparation of the DNA polymer.Digestion with restriction enzymes may be performed in an appropriatebuffer at a temperature of 20°-70° C., generally in a volume of 50 μl orless with 0.1-10 μg DNA.

The recombinant host cell is prepared, in accordance with the invention,by transforming a host cell with a replicable expression vector of theinvention under transforming conditions. Suitable transformingconditions are conventional and are described in, for example, Sambrooket al., cited above, or “DNA Cloning” Vol. II, D. M. Glover ed., IRLPress Ltd, 1985.

The choice of transforming conditions is determined by the host cell.Thus, a bacterial host such as E. coli, may be treated with a solutionof CaCl₂ (Cohen et al.,Proc. Nat. Acad. Sci., 1973, 69, 2110) or with asolution comprising a mixture of RbCl, MnCl₂, potassium acetate andglycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCland glycerol or by electroporation as for example described by Bio-RadLaboratories, Richmond, Calif., USA, manufacturers of an electroporator.Mammalian cells in culture may be transformed by calciumco-precipitation of the vector DNA onto the cells or by using cationicliposomes.

The invention also extends to a host cell transformed with a replicableexpression vector of the invention.

Culturing the transformed host cell under conditions permittingexpression of the DNA polymer is carried out conventionally, asdescribed in, for example, Sambrook et al., and “DNA Cloning” citedabove. Thus, preferably the cell is supplied with nutrient and culturedat a temperature below 45° C.

The proteion product is recovered by conventional methods according tothe host cell. Thus, where the host cell is bacterial such as E. coliand the protein is expressed intracellularly, it may be lysedphysically, chemically or enzymatically and the protein product isolatedfrom the resulting lysate. Where the host cell is mammamlian, theproduct is usually isolated from the nutrient medium.

Where the host cell is bacterial, such as E. coli, the product obtainedfrom the culture may require folding for optimum functional activity.This is most likely if the protein is expressed as inclusion bodies.There are a number of aspects of the isolation and folding process thatare regarded as important. In particular, the polypeptide is preferablypartially purified before folding, in order to minimise formation ofaggregates with contaminating proteins and minimise misfolding of thepolypeptide. Thus, the removal of contaminating E. coli proteins byspecifically isolating the inclusion bodies and the subsequentadditional purification prior to folding are important aspects of theprocedure.

The folding process is carried out in such a way as to minimiseaggregation of intermediate-folded states of the polypeptide. Thus,careful consideration needs to be given to, among others, the salt typeand concentration, temperature, protein concentration, redox bufferconcentrations and duration of folding. The exact condition for anygiven polypeptide generally cannot be predicted and must be determinedby experiment.

There are numerous methods available for the folding of proteins frominclusion bodies and these are known to the skilled worker in thisfield. The methods generally involve breaking all the disulphide bondsin the inclusion body, for example with 50 mM 2-mercaptoethanol, in thepresence of a high concentration of denaturant such as 8M urea or 6Mguanidine hydrochloride. The next step is to remove these agents toallow folding of the proteins to occur. Formation of the disulphidebridges requires an oxidising environment and this may be provided in anumber of ways, for example by air, or by incorporating a suitable redoxsystem, for example a mixture of reduced and oxidised glutathione.

Preferably, the inclusion body is solubilised using 8M urea, in thepresence of mercaptoethanol, and protein is folded, after initialremoval of contaminating proteins, by addition of cold buffer. Suitablebuffers may be identified using the techniques described in I.Dodd etat, ‘Perspectives in Protein Engineering and ComplementaryTechnologies’, Mayflower Publications, 66-69, 1995. A suitable bufferfor many of the SCR constructs described herein is 20 mM ethanolaminecontaining 1 mM reduced glutathione and 0.5 mM oxidised glutathione. Thefolding is preferably carried out at a temperature in the range 1 to 5°C. over a period of 1 to 4 days.

If any precipitation or aggregation is observed, the aggregated proteincan be removed in a number of ways, for example by centrifugation or bytreatment with precipitants such as ammonium sulphate. Where either ofthese procedures are adopted, monomeric polypeptide is the major solubleproduced.

If the bacterial cell secretes the protein, folding is not usuallynecessary.

The polypeptide portion of the derivative of the invention may include aC-terminal cysteine to facilitate post translational modification. Asoluble polypeptide including a C-terminal cysteine also forms part ofthe invention. Expression in a bacterial system is preferred forproteins of moderate size (up to −70 kDa) and with <−8 disulphidebridges. More complex proteins for which a free terminal Cys could causerefolding or stability problems may require stable expression inmammalian cell lines (especially CHO). This will also be needed if acarbohydrate membrane binding element is to be introducedpost-translationally. The use of insect cells infected with recombinantbaculovirus encoding the polypeptide portion is also a useful generalmethod for preparing more complex proteins and will be preferred when itis desired to carry out certain post-translational processes (such aspalmitoylation) biosynthetically (see for example. M. J.Page et alJ.Biol.Chem. 264, 19147-19154, 1989)

A preferred method of handling proteins C-terminally derivatised withcysteine is as a mixed disulphide with mercaptoethanol or glutathione oras the 2-nitro, 5-carboxyphenyl thio-derivative as generally describedbelow in Methods.

Peptide membrane binding elements may be prepared using standard solidstate synthesis such as the Merrifield method and this method can beadapted to incorporate required non-peptide membrane binding elementssuch as N-acyl groups derived from myristic or palmitic acids at the Nterminus of the peptide. In addition activation of an amino acid residuefor subsequent linkage to a protein can be achieved during chemicalsynthesis of such membrane binding elements. Examples of suchactivations include formation of the mixed 2-pyridyl disulphide with acysteine thiol or incorporation of an N-haloacetyl group. Both of thesegroups are capable of reaction with free thiols, through disulphideinterchange and alkylation, respectively. Peptides can optionally beprepared as the C-terminal amide and/or with a conventional N-terminalblocking group such as acetyl.

The invention also provides a peptidic membrane binding elementcomprising one or more derivatisations selected from:

a terminal cysteine residue optionally activated at the thiol group;

an N-haloacetyl group (where halo signifies chlorine, bromine or iodine)located at the N-terminus of the the peptide or at an E-amino group of alysine residue;

an amide group at the C-terminus;

an N-terminal blocking group; and

a fatty acid N-acyl group at the N-terminus or at an amino group of alysine residue.

Chemical bridging groups and reagents suitable for their formationinclude those described in EP0109653, EP0152736, EP0155388 andEP0284413, incorporated herein by reference. The bridging group isgenerally of the formula:

—A—R—B—  (I)

in which each of A and B, which may be the same or different, represents—CO—, —C(═NH₂ ⁺)—, maleimido, —S— or a bond and R is a bond or a linkinggroup containing one or more —(CH₂)— or meta-, ortho- or para-disubstituted phenyl units, preferably ortho or para, optionallytogether with a hydrophilic portion.

Where the polypeptide portion of the derivative of the invention and apeptidic membrane binding element both include a C-terminal cysteine thechemical bridging group will take the form —S—S—. The bridge isgenerated by conventional disulphide exchange chemistry, by activating athiol on one polypeptide and reacting the activated thiol with a freethiol on the other polypeptide. Such activation procedures make use ofdisulphides which form stable thiolate anions upon cleavage of the S—Slinkage and include reagents such as 2,2′ dithiopyridine and5,5′-dithio(2-nitrobenzoic acid, DTNB) which form intermediate mixeddisulphides capable of further reaction with thiols to give stabledisulphide linkages.

R may include moieties which interact with water to maintain the watersolubility of the linkage and suitable moieties include —CO—NH—,—CO—NMe—, —S—S—, —CH(OH)—, —SO₂—, —CO₂—, —(CH₂CH₂—O)_(m)— and —CH(COOH)—where m is an integer of 2 or more, or linear hydrophilic polymers suchas polyethylene glycol, polypropylene glycol, polyglycine, polyalanineor polysarcosine.

Other examples of R include —(CH₂)_(r)—, —(CH₂)_(p)—S—S—(CH₂)_(q)— and—(CH₂)_(p)—CH(OH)—CH(OH)—(CH₂)_(q)—, in which r is an integer of atleast 2, preferably at least 4 and p and q are independently integers ofat least 2

In a further aspect R may take the form —U—V—W— where U is(CH₂)₂CONH(CH₂)_(n) in which n is an integer of 3 to 8, V is O, S,NR_(a) or NR_(a)—NR_(a) where each R_(a) is H or C₁₋₆ allcyl, NH—O orO—NH, and W is benzyl substituted at the 2- or 4-position by the groupB. In a preferred embodiment R is (CH₂)₂CONH(CH₂)_(n)NH—(4-phenyl) wheren is an integer of 3 to 8. The bridging group of formula (I) may bederived from a linking agent of formula (II):

X—R₁—Y   (II)

in which R₁ is a bond or a linking group and X and Y are functionalgroups reactable with surface amino acid groups. preferably a lysine orcysteine group, the N-terminal amino group, a catalytic serine hydroxylor a protein attachment group, and X, R₁- and Y are chosen so as togenerate the required bridging group —A—R—B—.

Preferred agents are those where X and Y are different, known asheterobifunctional agents. Each end of the agent molecule is reacted inturn with each polypeptide to be linked in separate reactions. Examplesof heterobifunctional agents of formula (II) include:

N-succinimidyl 3-(2-pyridyldithio) propionate

succinimidyl 4-(N-maleimido) caproate

3-(2-pyridyl) methyl propionimidate hydrochloride

4′-amidinophenyl 4-N-[2-N-(3-[2-pyridyldithio]ethylcarbonyl)aminoethyl]aminobenzoate hydrochloride.

Other suitable agents are disclosed in EP0109653, EP0152736, EP0155388and EP0284413, in particular those of formula (II) in EP0155388 and(III) in EP0284413 incorporated herein by reference.

In each case Y is capable of reacting with a thiol group on apolypeptide, which may be a native thiol or one introduced as a proteinattachment group.

The protein attachment group is a functionality derived by modificationof a polypeptide or protein with a reagent specific for one or moreamino acid side chains, and which contains a group capable of reactingwith a cleavable section on the other polypeptide. An example of aprotein attachment group is a thiol group. An example of a cleavablesection is a disulphide bond. Alternatively the cleavable section maycomprise an a, dihydroxy function.

As an example, the introduction of a free thiol function by reaction ofa polypeptide with 2-iminothiolane, N-succinimidyl 3-(2-pyridyldithio)propionate (with subsequent reduction) or N-acetyl homocysteinethiolactone will permit coupling of the protein attachment group with athiol-reactive Y structure. Alternatively, the protein attachment groupcan contain a thiol-reactive entity such as the 6-maleimidohexyl groupor a 2-pyridyl-dithio group which can react with a free thiol in X.Preferably, the protein attachment group is derived from proteinmodifying agents such as 2-iminothiolane that react with lysine ε-aminogroups in proteins.

When X represents a group capable of reacting directly with the aminoacid side chain of a protein. it is preferably an N-succinimidyl group.When X represents a group capable of reacting with a protein attachmentgroup. it is preferably a pyridylthio group. When X represents a groupcapable of reacting with a catalytic serine hydroxyl it is preferably an4-amidinophenyl ester group optionally substituted by one or moreelectron withdrawing groups which increases the reactivity of the ester,of the kind contained in the compounds of formula (II) in EP0155388 and(III) in EP0284413.

In the above processes, modification of a polypeptide to introduce aprotein attachment group is preferably carried out in aqueous bufferedmedia at a pH between 3.0 and 9.0 depending on the reagent used. For apreferred reagent, 2-iminothiolane, the pH is preferably 6.5-8.5. Theconcentration of polypeptide is preferably high (>10 mg/ml) and themodifying reagent is used in a moderate (1.1- to 5-fold) molar excess,depending on the reactivity of the reagent. The temperature and durationof reaction are preferably in the range 0°-40° C. and 10 minutes to 7days. The extent of modification of the polypeptide may be determined byassaying for attachment groups introduced.

Such assays may be standard protein chemical techniques such astitration with 5,5′-dithiobis-(2-nitrobenzoic acid). Preferably, 0.5-3.0moles of protein attachment group will be introduced on average per moleof polypeptide. The modified polypeptide may be separated from excessmodifying agents by standard techniques such as dialysis,ultrafiltration, gel filtration and solvent or salt precipitation. Theintermediate material may be stored in frozen solution or lyophilised.

Where the linking agent of formula (II) contains an amidino phenyl estergroup X the agent is preferably first reacted with a polypeptide enzymevia the group X and the reaction is preferably carried out under theconditions described for the introduction of blocking groups in EuropeanPublished Patent Application No. 0,009,879. Having been freed of excessreagent by suitable techniques such as high performance size exclusionchromatography or diafiltration, the acylated enzyme may then be reactedwith the other polypeptide at a ratio of approximately 1:1 in anon-nucleophilic buffer at pH7.0-8.0 and 0°-30° C. for up to 6 h.However, it is preferable to conduct the coupling below 10° C.(preferably 0°-4° C.) in order to minimise the hydrolysis of theacylated enzyme.

Where a protein attachment group is introduced in this way, the bridginggroup (I) will be formed from a reaction of the linking agent (H) andthe protein attachment group.

The polypeptides to be linked are reacted separately with the linkingagent or the reagent for introducing a protein attachment group bytypically adding an excess of the reagent to the polypeptide, usually ina neutral or moderately alkaline buffer, and after reaction removing lowmolecular weight materials by gel filtration or dialysis. The preciseconditions of pH, temperature, buffer and reaction time will depend onthe nature of the reagent used and the polypeptide to be modified. Thepolypeptide linkage reaction is preferably carried out by mixing themodified polypeptides in neutral buffer in an equimolar ratio. Otherreaction conditions e.g. time and temperature, should be chosen toobtain the desired degree of linkage. If thiol exchange reactions areinvolved, the reaction should preferably be carried out under anatmosphere of nitrogen. Preferably, UV-active products are produced (egfrom the release of pyridine 2-thione from 2-pyridyl dithio derivatives)so that coupling can be monitored.

After the linkage reaction, the polypeptide conjugate can be isolated bya number of chromatographic procedures such as gel filtration,ion-exchange chromatography, affinity chromatography or hydrophobicinteraction chromatography. These procedures my be either low pressureor high performance variants.

The conjugate may be characterised by a number of techniques includinglow pressure or high performance gel filtration, SDS polyacrylamide gelelectrophoresis or isoelectric focussing.

Membrane binding elements which are fatty acid derivatives are attachedpost translationally to a peptidic membrane binding element, preferablyat the terminus of the polypetide chain. Preferably, where therecombinant polypeptide portion of the derivative of the inventioncontains the peptidic membrane binding element, it has a unique cysteinefor coupling to the fatty acid derivative. Where the recombinantpolypeptide has a cysteine residue, a thiol-derivative of the fatty acidis added to the refolded recombinant protein at a late stage inpurification (but not necessarily the final stage) and at a reagentconcentration preferably below the critical micelle concentration. Oneof the fatty acid derivative and the recombinant peptide will have thethiol group activated as described above for thiol interchangereactions. The fatty acid derivative is preferably a C₁₀₋₂₀ fatty acylderivative of an aminoC_(2,6)allrane thiol (optionally C-substituted)such as N-(2-myristoyl) aminoethanethiol or N-myristoyl L-cysteine andforms part of the invention.

Suitable examples of hydrophilic synthetic polymers includepolyethyleneglycol (PEG), preferably α,ω functionalised derivatives,more preferably α-amino, ω-carboxy-PEG of molecular weight between 400and 5000 daltons which are linked to the polypeptide for example bysolid-phase synthesis methods (amino group derivatisation) or bythiol-interchange chemistry.

Membrane binding elements derived from ligands of known integralmembrane proteins, either amino acid sequences or carbohydrates, may begenerated by post-translational modification using the glycosylationpathways of eukaryotic cells targeted to N-linked glycosylation sites inthe peptide sequence.

Convenient generic final stage purification strategies are hydrophobicinteraction chromatography (HIC) on C2-C8 media and cation exchangechromatography for separation of derivatised and underivatised proteinsinto which a hydrophobic-electrostatic switch combination has beeninserted.

The derivatives of this invention are preferably administered aspharmaceutical compositions.

Accordingly, the present invention also provides a pharmaceuticalcomposition comprising a derivative of the invention in combination witha pharmaceutically acceptable carrier.

The compositions according to the invention may be formulated inaccordance with routine procedures for administration by any route, suchas oral, topical, parenteral, sublingual or transdermal or byinhalation. The compositions may be in the form of tablets, capsules,powders, granules, lozenges, creams or liquid preparations, such as oralor sterile parenteral solutions or suspensions or in the form of aspray, aerosol or other conventional method for inhalation.

The topical formulations of the present invention may be presented as,for instance, ointments, creams or lotions, eye ointments and eye or eardrops, impregnated dressings and aerosols, and may contain appropriateconventional additives such as preservatives, solvents to assist drugpenetration and emollients in ointments and creams.

The formulations may also contain compatible conventional carriers, suchas cream or ointment bases and ethanol or oleyl alcohol for lotions.Such carriers may be present as from about 1% up to about 98% of theformulation. More usually they will form up to about 80% of theformulation.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrollidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; or acceptable wettingagents such as sodium lauryl sulphate. Tablets may also contain agentsfor the stablisation of polypeptide drugs against proteolysis andabsorbtion-enhancing agents for macromolecules. The tablets may becoated according to methods well known in normal pharmaceuticalpractice.

Suppositories will contain conventional suppository bases, e.g.cocoa-butter or other glyceride.

For parenteral administration, fluid unit dosage forms are preparedutilizing the compound and a sterile vehicle, water being preferred. Thecompound, depending on the vehicle and concentration used, is dissolvedin the vehicle. In preparing solutions the compound can be dissolved inwater for injection and filter sterilised before filling into a suitablevial or ampoule and sealing.

Parenteral formulations may include sustained-release systems such asencapsulation within microspheres of biodegradable polymers such aspoly-lactic co-glycolic acid.

Advantageously, agents such as a local anaesthetic, preservative andbuffering agents can be dissolved in the vehicle. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum. The dry lyophilized powder is thensealed in the vial and an accompanying vial of water for injection maybe supplied to reconstitute the liquid prior to use. Advantageously, asurfactant or wetting agent is included in the composition to facilitateuniform distribution of the compound.

Compositions of this invention may also suitably be presented foradministration to the respiratory tract as a snuff or an aerosol orsolution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase the particles of active compound suitably have diameters of lessthan 50 microns, preferably less than 10 microns for example diametersin the range of 1-50 microns, 1-10 microns or 1-5 microns. Whereappropriate, small amounts of anti-asthmatics and bronchodilators, forexample sympathomimetic amines such as isoprenaline, isoetharine,salbutamol, phenylephrine and ephedrine; xanthine derivatives such astheophylline and aminophylline and corticosteroids such as prednisoloneand adrenal stimulants such as ACTH may be included.

Microfine powder formulations may suitably be administered in an aerosolas a metered dose or by means of a suitable breath-activated device.

Suitable metered dose aerosol formulations comprise conventionalpropellants, cosolvents, such as ethanol, surfactants such as oleylalcohol, lubricants such as oleyl alcohol, desiccants such as calciumsulphate and density modifiers such as sodium chloride.

Suitable solutions for a nebulizer are isotonic sterilised solutions,optionally buffered, at for example between pH 4-7, containing up to20mg ml-¹ of compound but more generally 0.1 to 10 mg ml-¹, for use withstandard nebulisation equipment.

The quantity of material administered will depend upon the potency ofthe derivative and the nature of the complaint be decided according tothe circumstances by the physician supervising treatment. However, ingeneral, an effective amount of the polypeptide for the treatment of adisease or disorder is in the dose range of 0.01-100 mg/kg per day,preferably 0.1 mg-10 mg/kg per day, administered in up to five doses orby infusion.

No adverse toxicological effects are indicated with the compounds of theinvention within the above described dosage range.

The invention also provides a derivative of the invention for use as amedicament.

The invention further provides a method of treatment of disordersamenable to treatment by a soluble peptide which comprises administeringa soluble derivative of said soluble peptide according to the invention,and the use of a derivative of the invention for the preparation of amedicament for treatment of such disorders.

In one preferred aspect the present invention relates to derivatives foruse in the therapy of disorders involving complement activity andvarious inflammatory and immune disorders.

In this preferred aspect the soluble polypeptide which is derivatised inaccordance with the invention is a soluble complement inhibitor such asa soluble CR1 polypeptide fragment.

Constituting about 10% of the globulins in normal serum, the complementsystem is composed of many different proteins that are important in theimmune system's response to foreign antigens. The complement systembecomes activated when its primary components are cleaved and theproducts alone or with other proteins, activate additional complementproteins resulting in a proteolytic cascade. Activation of thecomplement system leads to a variety of responses including increasedvascular permeability, chemotaxis of phagocytic cells, activation ofinflammatory cells, opsonization of foreign particles, direct killing ofcells and tissue damage. Activation of the complement system may betriggered by antigen-antibody complexes (the classical pathway) or, forexample, by lipopolysaccharides present in cell walls of pathogenicbacteria (the alternative pathway).

Complement receptor type 1 (CR1) has been shown to be present on themembranes of erythrocytes, monocytes/macrophages, granulocytes, B cells,some T cells, splenic follicular dendritic cells. and glomerularpodocytes. CR1 binds to the complement components C3b and C4b and hasalso been referred to as the C3b/C4b receptor. The structuralorganisation and primary sequence of one allotype of CR1 is known(Klickstein petal, 1987, J. Exp. Med. 165:1095-1112, Klickstein et al.,1988, J. Exp. Med. 168:1699-1717; Hourcade et al., 1988, J. Exp. Med.168:1255-1270, WO 89/09220, WO 91/05047). It is composed of 30 shortconsensus repeats (SCRs) that each contain around 60-70 amino acids. Ineach SCR, around 29 of the average 65 amino acids are conserved. EachSCR has been proposed to form a three dimensional triple loop structurethrough disulphide linkages with the third and first and the fourth andsecond half-cystines in disulphide bonds. CR1 is further arranged as 4long homologous repeats (LHRs) of 7 SCRs each. Following a leadersequence, the CR1 molecule consists of the N-terminal LHR-A, the nexttwo repeats, LHR-B and LHR-C, and the most C-terminal LHR-D followed by2 additional SCRs, a 25 residue putative transmembrane region and a 43residue cytoplasmic tail.

Based on the mature CR1 molecule having a predicted N-terminal glutamineresidue, hereinafter designated as residue 1, the first four SCR domainsof LHR-A are defined herein as consisting of residues 2-58, 63-120,125-191 and 197-252, respectively, of mature CR1.

Several soluble fragments of CR₁ have been generated via recombinant DNAprocedures by eliminating the transmembrane region from the DNAs beingexpressed (WO 89/09220, WO 91/05047). The soluble CR1 fragments werefunctionally active, bound C3b andlor C4b and demonstrated Factor Icofactor activity depending upon the regions they contained. Suchconstructs inhibited in vitro complement-related functions such asneutrophil oxidative burst, complement mediated hemolysis, and C3a andC5a production. A particular soluble construct, sCR1/pBSCR1c, alsodemonstrated in vivo activity in a reversed passive Arthus reaction (WO89/09220, WO 91/05047; Yeh et al., 1991, J. Immunol. 146:250),suppressed post-ischemic myocardial inflammation and necrosis (WO89/09220, WO 91/05047; Weisman et al., Science, 1990, 249:146-1511;Dupe, R. et al. Thrombosis & Haemostasis (1991) 65(6) 695.) and extendedsurvival rates following transplantation (Pruitt & Bollinger, 1991, J.Surg. Res 50:350; Pruitt et al., 1991 Transplantation 52; 868).Furthermore, co-formulation of sCR1/pBSCR1c with panisoylated humanplasminogen-streptokinase-activator complex (APSAC) resulted in similaranti-haemolytic activity as sCR1 alone, indicating that the combinationof the complement inhibitor sCR1 with a thrombolytic agent was feasible(WO 91/05047).

The soluble CR1 polypeptide fragment encoded by sCR1/pBSCR1c, residues1-1929 of CR1, may be derivatised in accordance with the invention.

Soluble polypeptides corresponding to part of CR1 have been found topossess functional complement inhibitory, including anti-haemolytic,activity. WO94/00571 discloses soluble polypeptides comprising, insequence, one to four short consensus repeats (SCR) selected from SCR 1,2, 3 and 4 of long homologous repeat A (LHR-A) as the only structurallyand functionally intact SCR domains of CR1 and including at least SCR3.

In preferred aspects, the polypeptide comprises. in sequence, SCR 1, 2,3 and 4 of LHR-A or SCR 1, 2 and 3 of LHR-A as the only structurally andfunctionally intact SCR domains of CR1.

In one aspect, the polypeptides may be represented symbolically asfollows:

NH₂—V¹—SCR1—W¹—SCR2—X¹—SCR3—Y¹—OH   (A)

in which SCR1 represents residues 2-58 of mature CR1, SCR2 representsresidues 63-120 of mature CR1, SCR3 represents residues 125-191 ofmature CR1, and V¹, W¹, X¹ and Y¹ represent bonds or short linkingsequences of amino acids, preferably 1 to 5 residues in length and whichare preferably derived from native interdomain sequences in CR1.

In a preferred embodiment of formula (I), W¹, X¹ and Y¹ representresidues 59-62, 121-124 and 192-196, respectively, of mature CR1 and V¹represents residue 1 of mature CR1 optionally linked via its N-terminusto methionine.

In another aspect the polypeptides may be represented symbolically asfollows:

NH₂—V²—SCR1—W²—SCR2—X²—SCR3—Y²—SCR4—Z²OH   (B)

in which SCR1, SCR2 and SCR3 are as hereinbefore defined, SCR4represents residues 197-252 of mature CR1 and V², W², X², Y² and Z²represents bonds or short linking sequences of amino acids. preferably 1to 5 residues in length and which are preferably derived from nativeinterdomain sequences in CR1.

In preferred embodiments of formula (II), W², X², Y² and Z² representresidues 59-62, 121-124, 192-196, and residues 253 respectively, ofmature CR1 and V² represents residue 1 of mature CR1 optionally linkedvia its N-terminus to methionine.

In one particular embodiment of formula (B) arginine 253 is replaced byhistidine.

In the preferred embodiment of formula (B), residue 235 is arginine.

In one further aspect, the polypeptide may be represented symbolicallyas follows:

NH₂—X³—SCR3—Y³—OH   (C)

in which SCR3 is as hereinbefore defined and X³ and Y³ represent bondsor short linking sequences of amino acids, preferably 1 to 5 residues inlength and which are preferably derived from native interdomainsequences in CR1.

In a preferred embodiment of formula (C) X³ represents amino acids122-124 of mature CR1 optionally linked to methionine at its N-terminusand Y⁴ represents amino acids 192-196 of mature CR1.

In another further aspect, the polypeptide may be representedsymbolically as follows:

NH₂—X⁴—SCR3—Y⁴—SCR4—Z⁴—OH   (D)

in which SCR3 and SCR4 are as hereinbefore defined and X⁴, Y⁴ and Z⁴represent bonds or short linking sequences of amino acids, preferably 1to 5 residues in length and which are preferably derived from nativeinterdomain sequences in CR1.

In a preferred embodiment of formula (D) X⁴ represents amino acids122-124 of mature CR1 optionally linked to methionine at its N-terminusand Y⁴ and Z⁴ represent amino acids 192-196 and 253 respectively ofmature CR1.

The soluble CR1 polypeptide is derivatised in accordance with theinvention by any convenient strategy from those outlined above. In apreferred embodiment the soluble CR1 polypeptide consists of residues1-196 of CR1 and with an N-terminal methionine and the derivativecomprises a myristoyl group and one or more polypeptides sequenceselected from

DGPKKKKKKSPSKSSGC (SEQ ID NO: 36)

GSSKSPSKKKKKKPGDC (SEQ ID NO: 5)

CDGPKKKKKKSPSKSSK (SEQ ID NO: 18)

SKDGKKKKKKSKTKC (SEQ ID NO: 19)

CSAAPSSGFRILLLKV (SEQ ID NO: 20)

AAPSVIGFRILLLKVAGC (SEQ ID NO: 43)

and

DOPSEILRGDFSSC (SEQ ID NO: 44)

(N-terminus on left).

The soluble complement inhibitor, such as a soluble CR1 polypeptide,derivative of this invention is useful in the treatment of manycomplement-mediated or complement-related diseases and disordersincluding, but not limited to, those listed below.

Disease and Disorders Involving Complement Neurological Disorders

multiple sclerosis

stroke

Guillain Barré Syndrome

traumatic brain injury

Parkinson's disease

allergic encephalitis

Alzheimer's disease

Disorders of Inappropriate or Undesirable Complement Activation

haemodialysis complications

hyperacute allograft rejection

xenograft rejection

corneal graft rejection

interleukin-2 induced toxicity during IL-2 therapy

paroxysmal nocturnal haemoglobinuria

Inflammatory Disorders

inflammation of autoimmune diseases

Crohn's Disease

adult respiratory distress syndrome

thermal injury including burns or frostbite

uveitis

psoriasis

asthma

acute pancreatitis

Post-Ischemic Reperfusion Conditions

myocardial infarction

balloon angioplasty

atherosclerosis (cholesterol-induced) & restenosis

hypertension

post-pump syndrome in cardiopulmonary bypass or renal haemodialysis

renal ischemia

intestinal ischaemia

Infectious Diseases or Sepsis

multiple organ failure

septic shock

Immune Complex Disorders and Autoimmune Diseases

rheumatoid arthritis

systemic lupus erythematosus (SLE)

SLE nephritis

proliferative nephritis

glomerulonephritis

haemolytic anemia

myasthenia gravis

Reproductive Disorders

antibody- or complement-mediated infertility

Wound Healing

The present invention is also directed to a pharmaceutical compositionfor treating a disease or disorder associated with inflammation orinappropriate complement activation comprising a therapeuticallyeffective amount of a soluble complement inhibitor, such as a solubleCR1 polypeptide, derivative of this invention, and a pharmaceuticallyacceptable carrier or excipient.

The present invention also provides a method of treating a disease ordisorder associated with inflammation or inappropriate complementactivation comprising administering to a subject in need of suchtreatment a therapeutically effective amount of a soluble complementinhibitor, such as a soluble CR1 polypeptide, derivative of thisinvention.

In the above methods, the subject is preferably a human.

Further provided is the use of a soluble complement inhibitor, such as asoluble CR1 polypeptide, derivative of this invention in the manufactureof a medicament for the treatment of a disease or disorder associatedwith inflammation or inappropriate complement activation.

In order to inhibit complement activation and, at the same time, providethrombolytic therapy, the present invention provides compositions whichfurther comprise a therapeutically active amount of a thrombolyticagent. An effective amount of a thrombolytic agent is in the dose rangeof 0.01-10 mg/kg; preferably 0.1-5 mg/kg. Preferred thrombolytic agentsinclude, but are not limited to, streptokinase, human tissue typeplasminogen activator and urokinase molecules and derivatives, fragmentsor conjugates thereof. The thrombolytic agents may comprise one or morechains that may be fused or reversibly linked to other agents to formhybrid molecules (EP-A-0297882 and EP 155387), such as, for example,urokinase linked to plasmin (EP-A-0152736), a fibrinolytic enzyme linkedto a water-soluble polymer (EP-A-0183503). The thrombolytic agents mayalso comprise muteins of plasminogen activators (EP-A-0207589). In apreferred embodiment, the thrombolytic agent may comprise a reversiblyblocked in vitro fibrinolytic enzyme as described in U.S. Pat. No.4,285,932. A most preferred enzyme is the p-anisoylplasminogen-streptokinase activator complex, anistreplase as describedin U.S. Pat. No. 4,808,405 (Monk et al., 1987, Drugs 34:25-49).

Routes of administration for the individual or combined therapeuticcompositions of the present invention include standard routes, such as,for example, intravenous infusion or bolus injection. Active complementinhibitors and thrombolytic agents may be administered together orsequentially, in any order.

The present invention also provides a method for treating a thromboticcondition, in particular acute myocardial infarction, in a human ornon-human animal. This method comprises administering to a human oranimal in need of this treatment an effective amount of a solublecomplement inhibitor, such as a soluble CR1 polypeptide, derivativeaccording to this invention and an effective amount of a thrombolyticagent.

Also provided is the use of a soluble complement inhibitor, such as asoluble CR1 polypeptide, derivative of this invention and a thrombolyticagent in the manufacture of a medicament for the treatment of athrombotic condition in a human or animal. Such methods and uses may becarried out as described in WO 91/05047.

This invention further provides a method for treating adult respiratorydistress syndrome (ARDS) in a human or non-human animal. This methodcomprises administering to the patient an effective amount of a solublecomplement inhibitor, such as a soluble CR1 polypeptide, derivativeaccording to this invention.

The invention also provides a method of delaying hyperacute allograft orhyperacute xenograft rejection in a human or non-human animal whichreceives a transplant by administering an effective amount of a solublecomplement inhibitor, such as a soluble CR1 polypeptide, derivativeaccording to this invention. Such administration may be to the patientor by application to the transplant prior to implantation.

The invention yet further provides a method of treating wounds in ahuman or non-human animal by administering by either topical orparenteral e.g. intravenous routes, an effective amount of a solublecomplement inhibitor, such as a soluble CR1 polypeptide derivativeaccording to this invention.

In another preferred aspect the soluble polypeptide is a thrombolyticagent such as prourokinase, streptokinase, tissue-type plasminogenactivator or reteplase and the derivative of the invention is useful inthe treatment of thrombotic disorders such as acute myocardialinfarction. The invention thus provides a pharmaceutical composition fortreating thrombotic disorders comprising a therapeutically effectiveamount of a derivative of a thrombolytic agent according to theinvention, and a pharmaceutically acceptable carrier or excipient. Theinvention further provides a method of treatment of thrombotic disordersby administering an effective amount of a derivative of a thrombolyticagent according to the invention, and the use of such derivative in thepreparation of a medicament for the treatment of thrombotic disorders.

The following Methods and Examples illustrate the invention.

GENERAL METHODS USED IN EXAMPLES

(i) DNA Cleavage

Cleavage of DNA by restriction endonucleases was carried out accordingto the manufacturer's instructions using supplied buffers. Doubledigests were carried out simultaneously if the buffer conditions weresuitable for both enzymes. Otherwise double digests were carried outsequentially where the enzyme requiring the lowest salt condition wasadded first to the digest. Once the digest was complete the saltconcentration was altered and the second enzyme added.

(ii) DNA ligation

Ligations were carried out using T4 DNA ligase purchased from Promega,as described in Sambrook et al, (1989) Molecular Cloning: A LaboratoryManual 2nd Edition. Cold Spring Harbour Laboratory Press.

(iii) Plasmid isolation

Plasmid isolation was carried out by the alkaline lysis method describedin Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual 2ndEdition. Cold Spring Harbour Laboratory Press or by one of twocommercially available kits: the Promega WizardTM Plus Minipreps orQiagen Plasmid Maxi kit according to the manufacturer's instructions.

(iv) DNA fragment isolation

DNA fragments were excised from agarose gels and DNA extracted using oneof three commercially available kits: the QIAEX gel extraction kit orQiaquick gel extraction kit (QIAGEN Inc., USA), or GeneClean (Bio 101Inc, USA) according to the manufacturer's instructions.

(v) Introduction of DNA into E. coli

Plasmids were transformed into E. coli BL21(DE3) (Studier and Moffat,(1986), J. Mol. Biol 189:113), E. coli XLI-blue, BL21 (DE3) pLys-S orBLR (DE3) pLys-S that had been made competent using calcium chloride asdescribed in Sambrook et al., (1989). Cell lines were purchased asfrozen competent cultures from Stratagene. E. coli JM109 was purchasedas a frozen competent culture from Promega.

(vi) DNA sequencing

Plasmid DNA was sequenced on a Vistra DNA Labstation 625. The sequencingchemistry was performed using Amersham International's Thermo Sequenasefluorescent dye-terminator cycle sequencing kit′ (RPN 2435), inconjunction with their FMP fluorescent dye-terminator precipitation kit′(RPN 2433) according to the manufacturer's instructions.

The sequences produced by the above procedure were analysed by a PerkinElmer ABI Prism 377 DNA Sequencer. This is an electrophoretic techniqueusing 36 cm×0.2mm 4% acrylamide gels, the fluorescently labeled DNAfragments being detected by a charge coupled device camera according tothe manufacturer's instructions.

(vii) Production of oligonucleotides

Oligonucleotides were purchased from Cruachem.

(viii) pBROC413

The plasmid pT7-7 [Tabor, S (1990), Current Protocols in MolecularBiology, F. A. Ausubel, Brent. R. E. Kingston, D. D. Moore, J. G.Seidman, J. A. Smith, and K. Struhl,eds.] pp.16.2.1-16.2.11, GreenePublishing and Wiley-lnterscience,New York.] contains DNA correspondingto nucleotides 2065-4362 of pBR322 and like pBR322 can be mobilized by aconjugative plasmid in the presence of a third plasmid ColK. A mobilityprotein encoded by ColK acts on the nic site at nucleotide 2254 ofpBR322 initiating mobilization from this point. pT7-7 was digested withLspI and BglII and the protruding 5′ ends filled in with the Klenowfragment of DNA PolymeraseI. The plasmid DNA fragment was purified byagarose gel electrophoresis, the blunt ends ligated together andtransformed into E. coli DH1 by electroporation using a Bio-Rad GenePulser and following the manufacturers recommended conditions. Theresultant plasmid pBROC413 was identified by restriction enzyme analysisof plasmid DNA.

The deletion in pBROC413 from the LspI site immediately upstream of thef10 promoter to the BglII site at nucleotide 434 of pT7-7 deletes theDNA corresponding to nucleotides 2065-2297 of pBR322. The nic site andadjacent sequences are therefore deleted making pBROC413 nonmobilizable.

(ix) Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE)

SDS PAGE was carried out generally using the Novex system (BritishBiotechnology) according to the manufacturer's instructions. Prepackedgels of 4-20% acrylamide were used. Samples for electrophoresis,including protein molecular weight standards (for example LMW Kit,Pharmacia or Novex Mark 12) were usually diluted in1%(w/v)SDS-containing buffer (with or without 5%(vlv)2-mercaptoethanol), and left at room temperature for about 10 to 30 minbefore application to the gel.

(x) Reduction of disulphides and modification of thiols in proteins

There are a number of methods used for achieving the title goals. Thereason it may be necessary to carry out selective reduction ofdisulphides is that during the isolation and purification of multi-thiolproteins, in particular during refolding of fully denatured multi-thiolproteins, inappropriate disulphide pairing can occur. In addition, evenif correct disulphide paring does occur, it is possible that a freecysteine in the protein may become blocked, for example withglutathione. These derivatives are generally quite stable. In order tomake them more reactive, for example for subsequent conjugation toanother functional group, they need to be selectively reduced, with forexample dithiothreitol (DTT) or Tris (2-carboxyethyl) phosphine.HCl(TCEP) then optionally modified with a function which is moderatelyunstable. An example of the latter is Ellmans reagent (DTNB) which givesa mixed disulphide. In the case where treatment with DTNB is omitted,careful attention to experimental design is necessary to ensure thatdimerisation of the free thiol-containing protein is minimised.Reference to the term ‘selectively reduced’ above means that reactionconditions eg. duration, temperature, molar ratios of reactants have tobe carefully controlled so that reduction of disulphide bridges withinthe natural architecture of the protein is minimised. All the reagentsare commercially available eg. from Sigma or Pierce.

The following general examples illustrate the type of conditions thatmay be used and that are useful for the generation of free thiols andtheir optional modification. The specific reaction conditions to achieveoptimal thiol reduction and/or modification are ideally determined foreach protein batch.

TCEP may be prepared as a 20 mM solution in 50 mM Hepes (approx. pH 4.5)and may be stored at −40 degrees C. DTT may be prepared at 10 mM insodium phosphate pH 7.0 and may be stored at −40 degrees C. DTNB may beprepared at 10 mM in sodium phosphate pH 7.0 and may be stored at −40degrees C. All of the above reagents are typically used at molarequivalence or molar excess over protein concentration, the preciseconcentrations ideally identified experimentally. The duration and thetemperature of the reaction are similarly determined experimentally.Generally the duration would be in the range 1 to 24 hours and thetemperature would be in the range 2 to 30 degrees C. Excess reagent maybe conveniently removed by buffer exchange, for example using SephadexG25 or Sephadex G50. A suitable buffer is 0.1M sodium phosphate pH7.0 orthe solution may be left untreated.

EXAMPLES Example 1 Prepared a [N-(Myristoyl) 3-aminoethane thiol (MAET)

Myristoyl chloride (1.0 mmol) was added with vigorous stirring toice-cooled dry pyridine (1.0 ml), and followed immediately byN-hydroxysaccinimide (1.5 mmol). The mixture was stirred for 4 h atambient temperature (−23° C.). 2-aminoethanethiol free base (1.1 mmol)was added as solid to the mixture and allowed to react for 6 h atambient temperature, followed by 3 days at 4° C. The product was treatedwith water (5 ml), stirred for 1 h at ambient and filtered, washing withcold water. The white solid was dissolved in dimethylsulphoxide andreprecipitated with water and then vacuum dried over phosphorouspentoxide. The final yield was 0.21 g (−70%). Thiol titration usingEllman's reagent indicated that the product contained −45% free thiol.

Example 2 Synthesis of Myristoyl/Electrostatic Switch Peptide Reagent 1(MSWP-1) (SEQ ID NO:27)

N-(Myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-2-Thiopyridyl)Cys-NH₂

The peptide:

Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-Cys-NH₂(SEQ ID NO:5) was prepared using solid phase synthesis via the generalFmoc/tBu strategy developed by Sheppard and Atherton (E. Atherton and R.C. Sheppard, Solid Phase Synthesis, IRL Press, Oxford, 1989).Kieselguhr-supported polydimethylacrylamide resin (Macrosorb 100) wasused as the solid support and was derivatised with ethylene diamine.

Coupling reactions were carried out using N-α-Fmoc protected reagentspreactivated with N,N′-diisopropylcarbodiimide/N-hydroxybenzotriazole(in 4-fold molar excess) with bromophenol blue monitoring. Fmoccleavages used 20% piperidine in DMF. Reactions to assemble the peptidechain were carried out by repeated cycles of coupling and deprotectionincluding the attachment of the modified Rink linkage reagent(p-[(R,S)-α-[1-(9H-fluoreny-9-yl-methoxyformamido]2,4dimethoxybenzyl]-phenoxyaceticacid) designed to yield a C-terminal amide on final cleavage. The sidechain functionalities of the individual amino-acids were protected asfollows:

Ser(tButyl), Lys (Boc), Asp (O-tButyl), Cys (Trityl).

On completion of the peptide assembly and with the peptide stillattached to the resin, the myristoyl group was attached to the aminogroup of the N terminal glycine by direct coupling of myristic acid bythe same activation procedure. This modified peptide was then cleavedfrom the resin and the side-chain protecting groups removed at the sametime by treatment with trifluoracetic acid containing 2.5% water and2.5% triisopropyl silane.

The crude product was treated with 2,2′ dithiopyridine in 0.01M ammoniumacetate solution at pH 8-9 for approx. 2 h, then acidified with aceticacid and purified by preparative high performance liquid chromatography(HPLC) in 0.1% trifluoracetic acid (TFA)/water and 0.1% TFA/acetonitrileas gradient component. After lyophilisation, the peptide was a whiteamorphoes powder, soluble to at least 10 mg/ml in dimethylsulphoxide.Fast atom bombardment mass spectrometry gave main peaks at m/e 2107.8,2129,7 and 2145.8, corresponding to the monoprotonated, monosodiated andmonopotassiated molecular ions of the peptide. The 2-thiopyridyl contentof the peptide was measured by dissolving is to around 0.03 mM to 0.2 mMin 0.1M Sodium Borate pH 8.0 and reducing by addition of dithiothreitolto 5 mM. The change in optical density at 343 nm was used to calculatethe amount of pyridine 2-thione released using an extinction coefficientat this wavelength of 8080 cm⁻¹ M⁻¹. This indicated that the peptidecontent was approximately 60% of the dry weight.

Example 3 Synthesis of Myristory/Electrostatic Switch Peptide Reagent 2(MSWF-2) (SEQ ID NO:28)

N-methyl-Cys(3-thiopyridyl)Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Prop-SerLys-Ser-Ser-Lys-(e-N-(Myristoyl))-NH₂

The peptide:

Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Pro-Ser-Lys-Ser-Ser-Lys-NH₂(SEQ ID NO:18) was prepared by solid-phase synthesis using the generalmethod described in Example 2 and with the following variations:

a. The C-terminal lysine was protected by alkylation with the 4-methyltrityl (MTT) group; all other lysines were N-ε protected with t-Bocgroup

b. MTT was removed with 1% v/v trifluoracetic acid in dichloromethaneand the resulting unique free amino group derivatised with myristic acidprior to deprotection of the other lysines (as described in Example 2).

The N-terminus was acetylated with acetic anhydride upon completion ofthe peptide chain assembly. Generation of the 2-pyridyldithiocysteinemoiety was by reaction of the deprotected peptide with2,2′-dithiopyridine as described above. The product was purified asdescribed in Example 2. Fast-atom bombardment mass spectrometry gave amolecular ion peak at 2221.3 (cf. 2220.3 for the monoprotonatedtheoretical mass).

Amino-acid Analysis: Asx Ser Gly Pro Theory: 1.0 4.0 1.0 2.0 Found 0.973.53 1.15 1.88 (Asx = Asn or Asp)

Amino-acid analysis indicated a net peptide content by weight of 68.7%.The 2-pyridyl disulphide content was approximately 60% by weight usingthe method of Example 2.

Example 4 Synthesis of Myristoyl/Electrostatic Switch Peptide Reagent 5(MSWP-3) (SEQ ID NO:29)

N-(Myristoyl)-Ser-Lys-Asp-Gly-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Lys-Thr-Lys-(S-3-Thiopyridyl)Cys-NH₂

The peptide:

Ser-Lys-Asp-Gly-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Lys-Thr-Lys-Cys (SEQ IDNO:19) was prepared using the general solid-phase synthesis protocol ofExample 2. Myristolyation, C-terminal amidation and derivatisation ofthe Cys residue were performed as described in Example 2. Afterpurification, mass spectrometry gave the major peak at 2040.5,corresponding to a monoprotonated form (Theory: 2039.5)

Amino-acid analysis: Asx Ser Gly Thr Lys Theory: 1 2 1 1 9 Found: 1.022.04 1.14 1.06 8.85

The peptide content was about 56% by weight

Example 5 Synthesis of T-Cell Targeting Peptide Reagent 1 (TCTP-1) (SEQID NO:30)

N-ethyl-(2-thiopyethyl)CysSer-Ala-Ala-Pro-Ser-Ser-Gly-Pho-Arg-Ile-Leu-Leu-Leu-Lys-Val-CONH(CH₃)₃CH₅

The peptide:

(Cys-Ser-Ala-Ala-Pro-Ser-Ser-Gly-Phe-Arg-Ile-Leu-Leu-Leu-Lys-Val (SEQ IDNO:20) was prepared using the general solid-phase methodology of Example2 and N-acetylated as in Example 3. The C-terminal was derivatized usingn-decylamine in place of the Rink reagent. Mass spectrometry of thepurified peptide gave a major peak at 1952.3 corresponding to amonoprotonated molecular in (Theory: 1951.1.) An ion at 1843.3 was alsoobserved, this is believed to correspond to loss of the thiopyridylgroup in the spectrophotometer.

Amino-acid analysis: Ser Gly Arg Ala Pro Val Ile Phe Leu Lys Theory: 3  1   1   2   1   1   1   1   3   1   Found: 2.95 1.10 1.10 2.11 1.04 0.600.92 1.00 3.03 1.03

The peptide content by weight was 53%

Example 6 Expression and isolation of [SCR1-3]-Cys (SEQ ID NO:6)

(a) Construction of plasmid pDB1030 encoding [SCR 1-3]-Cys

The plasmid coding for SCR1-3 of LHR-A of CR1, pDB1013-5 (patentapplication WO 94/00571) was digested with restriction endonucleasesEcoRl and HindIII and the 2.2 kB plasmid band was isolated from anagarose gel using a Qiagen Qisex DNA extraction kit according to themanufacturer's instructions. This is fragment 1. A second batch ofpDB1013-5 was digested with Bml and EcoRI and the 196 bp band wasextracted from agarose as above. This is fragment 2. Twooligonucleotides, SEQ ID No. 1 and SEQ ID No. 2, were annealed to give afinal DNA concentration of 100 pmoles/ul. The annealed oligo has aBml/EcoRI overhang and duplicates the sequence at the 3′ end ofpDB-1013-5 but in addition contains a codon coding for cysteine justbefore the stop codon. This is fragment 3.

Fragments 1,2 and 3 were ligated with T4 DNA ligase in a single reactionto give pDB1030. The ligated plasmid was transformed into competent E.coli IM109 purchased from Promega. Resulting colonies were analysed byrestriction endonuclease digestion and DNA sequencing confirmed that theencoded amino acid sequence of SCR(1-3) (SEQ ID No.27 of WO 94/00571)had been altered by a single C-terminal cysteine residue to give SEQ IDNo.6.

(b) Expression of [SCR1-3]-Cys from pDB1030

pD1030 was transformed into calcium chloride competent E. coli BL21(DE3)and resultant colonies were isolated and checked for plasmid content. Toexpress protein from pDB1030 in E. coli BL21 (DE3), a single colony wasinoculated into 10 ml LB-phosphate media (20 g/L tryptcine, 15 g/L yeastextract, 0.8 g/L NaCl, 0.2 g/L Na₂HPO4-0.1g/L KH₂PO₄) containing 50ug/ml ampicillin. The culture was grown for 6 hours at 37° C. 230 r.p.m.before being used to inoculate 100 ml of the name media containing 50ug/ml ampicillin. Growth was under the same conditions overnight. 25 mlof each culture were then used to isoculate 600 ml of the same mediawith 50 ug/ml ampicillin in 3 L erlenmeyer flasks. Cells were grown toan OD of 0.8-1.0 at A₆₀₀ nm. IFTG (isopropyl B-D galactopyranoside) wasadded to a final concentration of 1 mM and cells allowed to continuegrowth for a further 3-4 hours before harvesting by centrifugation at8000 g/10 min. Pellet from 2L of culture was stored at −80° C.

(e) Isolation, refolding, purification and formulation of [SCR1-3]-Cys

The methods described are essentially those detailed in Dodd L et al(1995) Protein Expression and Purification 6 727-736.

I) Isolation of Solubilised Inclusion Bodies

The frozen cell pellet of E. coli BL21(DE3) (pDB1030) was resuspended in50 mM Tris/50 mM NaCl/1 mM EDTA/0.1 mM PMSF pH 8.0 at a ratio of 33 mlfor each Here of culture pellet. The suspension was transferred to aglass beaker surrounded by ion and sonicated (Heat systems—UltrasonicsW380; 50×50% pulse, pulse time=5 sec.) for typically 3-6 minutes. Thedisrupted pellet was then frozen and stored at −80° C. Approx. 2 weekslater the sonicate was thawed and centrifuged at approx. 8000 g for 20min. The pellet was resuspended in 20 mM Tris/8M urea/1 mM EDTA/50 mM2-mercaptoethanol pH 8.5 (200 mL) at room temperature by vigorousswirling, then left for 1 h at room temperature followed by overnight at4° C.

II) Initial Purification using SP-Sepharose

To the viscous solution was added SP-Sepharose FF (approx. 30 g wetweight) this had been water washed and suction-dried. The mixture wasswirled vigorously and left static for 1-2 h at room temperature. Thesupernatant was decanted, sampled and discarded. The remaining slurrywas resuspended to a uniform suspension had poured into a glass jacketand allowed to settle into a packed bed. The columns was equilibratedwith 0.02M Tris/8M urea/0.05M 2-mercaptoethanol/0.001M EDTA pH 8.5 at 4°C. When the A₂₈₀ of the eluate had stabilised at baseline, the bufferwas changed to equilibration buffer additionally containing 1M NaCl. Asingle A₂₈₀ peak was eluted by the 1M NaCl-containing buffer; the volumewas approx. 50 ml. The protein concentration of the solution wasestimated by A₂₈₀ determination, using a molar extinction coefficient of25000 cm⁻¹ of a sample that had been buffer-exchanged (Sephadex (325)into 50 mM formic acid. This showed the product had a proteinconcentration of 1.6 mg/ml. The solution was stored at −40° C.

III) Folding and Further Processing

25 ml of the SP-Sepharose-purified product was added gradually over a 1min period to 780 ml freshly prepared, cold 0.02M ethanolamide/1 mM EDTAwith continuous swirling, and left static for 1 h/4° C. Reducedglutathione (GSH) was added to 1 mM and xidised glutathione (GSSG) wasadded to 0.5 mM. The solution was clear and was left static approx 2-3°C. for 3 d. The solution was then ultrafiltered using a YM10 membrane toa final retentate volume of about 35 ml: the retentate was slightlycloudy and had the appearance of a translucent solution. It was storedfor 12 days at 4° C. It was then spun at 30,000 g for 15 mins and thesupernatant mixed with 9 vol. 0.1M NaH₂PO₄/1M (NH₄)₂SO₄ pH 7.0 (BufferA) at room temperature and immediately centrifuged at 3000 rpm for 15min. The supernatant was ultrafiltered (YM10) to about 4 ml and thenbuffer-exchanged into 0.1M sodium phosphate pH 7.0 (5.0 ml); thissolution had a protein concentration of 1.7 mg/ml by A280 analysis. Itwas treated with dithio bis nitrobenzoic acid (DTNB) (8-fold molarexcess) for 30 min at room temperature. Free thiol content based on A412measurement and an extinction coefficient (for the fine thiombrobenzoateion) of 13,600 was 6 ubl equivalent to only about 10% derivatisation togive Product A. The majority of the product was believed to be[SCR1-3]-Cys where the free C-terminal thiol was blocked by reactionwith glutathione or 2-mercaptoethanol during the refolding stage.

(d) Alternative method for isolation, refolding, purification andformulation of [SCR1-3]-Cys

The method was similar to that described above, except that it moreclosely followed the procedures described in Dodd et al (op cit.).Notably, the ultrafiltered retentate post refolding was immediatelytreated with ammonium sulphate followed by clarification bycentrifugation and Butyl Trycpearl chromatography. The resultingA280-absorbing fractions that eluted at about 0.2 to 0.4M ammoniumsulphate were pooled and regarded as Product B. Starting with a nominal100 mg of fully reduced SCR1-3/cys, Product B contained 17 mg. Theproduct contained one major species by non-reduced SDS PAGE with anestimated purity of >90% and an apparent molecular weight of 21,000. Onthe basis of studies with similarly produced preparations it wasbelieved to be the S-glutathione and/or S-mercaptoethanol derivatisedform of the parent protein, although at least some batches produced in asimilar way or stored for a period of time might exist as the freecysteine variant. The product also contained a polypeptide with asapparent molecular weight of about 40,000. On the basis of studies withsimilar batches of protein enriched in this species it was identified asthe dimer of [SCR1-3]-Cys.

Example 7 Expression and isolation of SCR1-3/switch fusion (SEQ ID NO:7)

N₂N-[SCR1-3]-Asp-Cly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Pro-Ser-Lys-Ser-Ser-Gly-Cys-OH

(a) Construction of plasmid pDB1031 encoding SCR1-3/switch

Fragment 1 and fragment 2 of pDB1013-5 were the same as Example 6 above.Two digonucleotides, SEQ ID No. 3 and SEQ ID No. 4, prepared byCroachern were annealed to give a final DNA concentration of 100pmoles/ul. The annealed oligo has an Banl/EcoRl overhang and duplicatesthe sequence at the 3′ end of pDB1013-5 but in addition contains 17additional codons coding for DGPKKKKKKSPSKSSGC (SEQ ID NO:36) justbefore the stop codon. This is fragment 4.

Fragments 1, 2 and 4 wee ligated with T4 DNA ligase in a single reactionto give pDB1031. The ligated plasmid was transformed into competant E.coli JM109. Resulting colonies were analysed by restriction endonucleasedigestion and DNA sequencing confirmed that the encoded amino acidsequence of SCR1-3 (SEQ ID 27 of WO 94/00571) had been altered by Cterminal addition of amino acids DGPKKKKKKSPSKSSGC (SEQ ID NO:36) togive SEQ ID NO:7.

(b) Expression of SCR1-3/switch from pDB1031

pDB1031 was transformed into calcium chloride competent E. coli(BL21(DE3) and resultant colonies were isolated and checked for plasmidcontent. To express protein from pDB1031 in E. coli BL21(DE3), a singlecolony was inoculated into 10 ml LB-phosphate media (20 g/L. trypione,15 g/L yeast extract. 0.8 g/L NaCl, 0.2 g/L. Na₂HPO₄, 0.1 g/L KH₂PO₄)containing 50 ug/ml ampicillin. The culture was grown for 6 hours at 37°C. 230 r.p.m. before being used to inoculate 100 ml of the same mediacontaining 50 ug/ml ampicillin. Growth was under the same conditionsovernight. 25 ml of each culture were then used to inoculate 600 ml ofthe same media with 50 ug/ml ampicillin in 3 L erlenmeyer flasks. Cellswere grown to an OD of 0.8-1.0 at A₆₀₀ nm. IPTG (isopropyl D-Dgalactopyranoside) was added to a final concentration of 1 mM and cellsallowed to continue growth for a further 3-4 hours before harvesting bycentrifugation at 8000 g/10 min. The cell pellet was frozen at −40degrees C.

(c) Isolation, refolding, purification and formulation of SCR1-3/switch

The methods described are essentially those detailed in Dodd I. et al.(1995) Protein Expression and Purification 6 727-736, with somemodifications.

I) Isolation of solubilised inclusion bodies

The frozen cell pellet of E. coli BL21(DE3) (pDB103)) was thawed andresuspended in 50 mM Tris/50 mM NaCl/1 mM EDTA/0.1 mM PMSF pH 8.0 at aratio of 33 ml for each liter of culture pellet. The suspension wastransferred to a glass beaker surrounded by ice and sonicated (Heatsystems—Ultrasonics W380; 50×50% pulse, pulse time=5 sec.) for typically3-6 mixtures. The disrupted pellet was then frozen and stored at −80° C.Approx. 1 d laser the sonicate was thawed and centrifuged at approx.8000 g for 20 min. The pellet was resuspended is 20 mM Tris/8M urea/1 mMEDTA/50 mM 2-mercaptoethanol pH 8.5 (240 ml) at room temperature byvigorous swirling, then left for 1 h at room temperature followed by 5days at 4° C.

II) Preliminary Purification using SP-Sepharose

To the viscous solution was added SP-Sepharose FF (approx. 30 g wetweight) that had been water washed and suction dried. The mixture wasswirled vigorously and left static for approx. 2 h at room temperature.The supernatant was decanted, sampled and discarded. The remainingslurry was resuspended to a uniform suspension and poured into a glassjacket and allowed to settle into a packed bed. The column wasequilibrated with 0.02M Tris/8M urea/0.05M 2-mercaptoethanol/0.001 MEDTA pH 8.5 at 4° C. When the A₂₈₀ of the eluate had stabilised atbaseline, the buffer was changed to equilibration buffer additionallycontaining 1M NaCl. A single A₂₈₀ peak was eluted by the 1MNaCl-containing buffer; the volume was approx. 50 ml. The proteinconcentration of the solution was estimated by A₂₈₀ determination, usinga molar extinction coefficient of 25000 cm⁻¹, of a sample that had beenbuffer-exchanged (Sephadex G25) into 50 mM formic acid. This showed theproduct had a protein concentration of 2.8 mg/ml. Analysis by SDSPAGE/main showed a major band (approx. 80%) at about 23,000Da. Thesolution was stored at −40° C.

III) Folding and Further Processing

14 ml of the SP-Sepharose-purified product was added gradually over a 1min period to 430 ml freshly prepared, cold 0.05M Hepes/2M sodiumchloride/1 mM EDTA pH 8.0 with continuous swirling, and left static for1 h/4° C. Reduced glutathione (GSH) was added to 1 mM and oxidizedglutathione (GSSG) was added to 0.5 mM. The solution was clear and wasleft static approx. 2-3° C. for 3 d. The solution was then ultrafilteredusing a YM10 membrane to a final retentate volume of about 34 ml; theretentate was slightly cloudy. It was then spun at 25 000 g for 15 minsand the supernatant buffer-exchanged into 0.1M sodium phosphate pH 7.0(46 ml). This fraction contained 2 mg of protein on the basis of an A280determination. The solution was mixed with DTNB (20 mM; 0.65 ml) for 20min at 4 degrees C. and then ultrafiltered to 2.4 ml. This retentate wasbuffer-changed into 0.1M sodium phosphate pH 7.0 (3.0 ml) and stored at−40 degrees C. Absorbance measurements at 412 mm on the solution priorto ultrafiltration suggested 25% derivatisation with DTNB.

(d) Alternative isolation, refolding, purification for formulation[SCR1-3]switch

The method was similar to that described in (c) above, except thatfollowing the ultrafiltration step after refolding it more closelyfollowed the procedures described in Dodd et al. (op cit.). Notably, theultrafiltered retentate post refolding was immediately treated withammonium sulphate followed by clarification by centrifugation and ButylSepharose chromatography. The resulting A280-absorbing fractions thateluted at about 0.2 to 0.4M ammonium sulphate were pooled and regardedas initial product. Additional treatment with TCEP essentially as above,followed by DTNB yielded a final product at 10 nM final proteinconcentrations. The final product contained one major species bynon-reduced SDS PAGE with an estimated porky of >90% and an apparentmolecular weight of 23,000 and contained about 2 moles TNB per mole ofprotein.

Example 8 Preparation of [SCR1-3]-Cys-S-S-[MSWP-1] (This sequence is aconjugate of SEQ ID NO:6 and the base peptide of SEQ ID NO:5).

(a) Product A of Example 6(c) (1.5 ml) was treated with dithiothreitol(30 ul of 0.5M in water, final concentration 10 mM) for 60 min at 4° C.to give the free peptide SEQ ID NO 6. The yellow solution was gelfiltered at 4° C. on a small column of Sephadex G-25 (PD-10, Pharmacia)into 0.05M Hepes HCl buffer pH 7.5 (3.0 ml). The slightly cloudysolution was mixed with a solution of MSWP-1 (Example 2) (3.8 mMdithiopyridyl equivalents, 150 ul) to a final concentration of 0.18 mM(−8 molar equivalents). The mixture was held for 2 h on ice and then gelfiltered as before but using 2 PD10 columns (1.5 ml applied, 3.2 mleluted). The final eluate was not cloudy and was stored frozen at −70°C. in aliquots of 0.4 ml.

(b) [SCR1-3]-Cys protein product B described in Example 6(d) (1.5 ml; 31mM protein) was mixed with TCEP (20 mM; 0.007 ml) and incubated at roomtemperature for 23 h to give the free protein SEQ ID NO:6, MSWP-1(Example 2) (10 mM; 0.093 ml) was added and the solution incubated for afurther 4 h. 0.75 ml of the final solution was buffer-exchanged into 50mM formic acid and aliquots left in solution or lyophilised. The productwas >80% pure by SDS PAGE and had an apparent molecular weight of23,000, clearly shifted from the original parent molecular weight of 21000. The lyophilised was easily soluble in 50 mM formic acid at anestimated protein concentration of 2 mg/ml.

(c) [SCR1-3]-Cys protein product B described in Example 6(d) (21.6 ml;31 uM protein) was mixed with TCEP (20 mM; 0.1 ml) and incubated at roomtemperature for 22 h to give the free protein SEQ ID NO 6. MSWP-1 (20 mMin 0.1M sodium phosphate pH 7.0; 0.67 ml) was added and the solutionincubated for a further 4 h. All 22 ml was buffer-exchanged into 50 mMformic acid using Sephades G50 (Vt 160 ml). Three A280 peaks wereobtained. The first one, eluting at volume 56-106 ml, was the tidecompound according to SDS PAGE analysis. The fraction was aliquoted andaliquots stored at −40 degrees C. or lyophilised. Amino acid analysis ofthe pro-lyophilisation solution indicated a protein concentration of0.42 mg/ml. A280 (1 cm path length) was 0.44. C8 reverse phase HPLC andSDS PAGE both indicated a purity of approx 80%. The latter techniqueshowed the major based had an apparent molecular weight of 23 000. Thelatter technique showed the major band had an apparent molecular weightof 23 000, clearly shifted from the original percent molecular weight of21,000; on reduction the 23,000 band shifted to two bands with molecularweights of approx 21,000 and approx 5,000. The lyophilisate was easilysoluble in 50 mM formic acid or in PBS‘A’ (Dalbecco) at a proteinconcentration of 6 mg/ml.

(d) [SCR1-3]-Cys-S-S-[MSWP-1] from (c) was divided into 0.3 ml aliquotsand freeze-dried. Individual aliquots were resolubilised in 50 mM formicacid (0.3 ml or 0.039 ml).

Example 9 Preparation of [SCR1-3/switch fusion]disulphide linked to[MAET] (SEQ ID NO:31)

Title compound can be synthesised using TNB-activated SCR1-3/switch (SEQID NO:7) prepared as in Example 7(d). The TNB-activated SCR1-3/switch ismixed with a molar excess of MAET (Example 1), which might be typicallymade up at 2.0 mg/ml in DMSO, equivalent to about 3 mM free thiol.Typical reaction conditions would be 1 to 4 hours at room temperature orovernight at 4 degrees C. using a protein concentration of 1 to 100 uM.The reaction may be monitored by checking the generation of yellowcolour, which is caused by the release of free TNB ion. Ones thereaction is complete the solution may be buffer exchanged into asuitable buffer, for example 0.1M sodium phosphate pH 7.0, and stored at−40 degrees C. until required.

Example 10 Preparation of [SCR1-3/switch fusion] disulphide linked to[MSWP-1] (This sequence is a conjugate of SEQ ID NO: 7 and the basepeptide of SEQ ID NO:5).

Method (a)

0.02 ml of MSWP-1 (Example 2, 10 mM in 0.1M sodium phosphate pH 7.0) wasmixed with 0.005 ml of TCEP (20 mM in 50 mM Hepes) and left for 10 minat room temperature. The resultant solution was Solution A containingthe myristoylated peptide of SEQ ID NO:5. TNB-activated SCR1-3/switch(SEQ ID NO:7) prepared in a similar way to that described in Example7(c) (0.3 ml; 15 uM in 0.1M sodium phosphate pH 7.0) was mixed with0.0056 ml of Solution A to give a theoretical MSWP-SH molar excess offive-fold over protein. The mixture was left for 4H at room temperaturefollowed by 18 h at 4 degrees C. Analysis by SDS PAGE followed byprotein staining indicated one major band at apparent M_(f) 23 K,corresponding to unreacted protein, and a minor band at apparent M_(f)26K, corresponding to title protein.

Method (b)

TNB-activated SCR1-3/switch product (SEQ ID NO:7) (10 uM; 0.43 ml)prepared in a similar way to that described in Example 7(d) was mixedwith TCEP (5 mM; 0.026 ml) and incubated for 17 h at room temperature toyield the free fusion protein SEQ ID NO:7. MSWP-1 (10 mM; 0.0086 ml) wasadded and incubation was continued for a further 4 h. Small particles orcrystals were present in the solution, but it was otherwise clear. Theparticulate solution was buffer-exchanged into 50 mM formic acid (1.0ml), aliquoted and frozen. Analysis by SDS PAGE under non-reducingconditions showed a number of bands, which included a species with asapparent molecular weight of 25,000 —the target species.

Example 11 Preparation of [SCR1: 1-1929]-Cys-S-S-[MSWP-1] (This sequenceis a conjugate of SEQ ID NO: 52 and the base peptide of SEQ ID NO:5).

Human complement receptor 1 (CR1, CD35) is a known regulator ofcomplement activation which has been produced in a recombinant solubleform containing all of the extracellular SCR domains of a major naturalallotype (Fearon et al. WO 89/09220, WO 91/05047). This form (sCR1) hasbeen expressed as an active protein in Chinese Hamster Ovary (CHO)cells. Mutagenesis of the DNA sequence immediately downstream of thecodon for Cys-1924 is performed to generate a new C-terminal cysteineresidue.

A suitable example of a modified terminus of the cDNA sequence of sCR1is as follows:

(5909) BalI(5914)

. . . ..CCT CTG GCC AAA TGT ACC TCT CGT GCA CAT TGC TGA (SEQ ID NO:45)the codon Asp-1930 in CR1 is replaced by that for a Cysteine (followedby a stop codon to generate a soluble protein) through ligation of amodified oligonucleotide to the unique BalI restriction endonucleasesite at position 5914 (numbering from Fearon et al., 1989, 1991).

Expression of this modified cDNA in CHO cells and isolation of theproduct by standard chromatographic procedures generates a modified sCF1protein which can be treated as in Example 8(a), (b) or (c) to couple itto MSWP-1 (Example 2) to yield the title compound.

Example 12 Preparation of [SCR1-3]-Cys-S-S-[MSWP-2] (This sequence is aconjugate of SEQ ID NO:6 and the base peptide of SEQ ID NO:18).

[SCR1-3]-Cys protein (SEQ ID NO:6) prepared in a similar way to thatdescribed in Example 6(d) (46 uM protein; 0.20 ml) was mixed with TCEP(5 mM; 0.0054 ml) was incubated at room temperature for approx. 20 h.0.05 ml of this solution was mixed with 0.025 ml of 0.1M ethanolamineand 0.003 ml of MSWP-2 (see Example 3; 5 mM in DMSO;); the solution wasincubated for a further 3 h at room temperature. SDS PAGE analysisshowed the major band in the preparation had an apparent molecularweight of 23,000, clearly shifted from the original parent molecularweight of 21,000. The purity of the target protein was estimated fromthe SDS PAGE gel to be approx 80%.

Example 13 Preparation of [SCR1:1-3]-Cys-S-S-[MSWP-3] (This sequence isa conjugate of SEQ ID NO:6 and the base peptide of SEQ ID NO:19).

[SCR1-3]-Cys protein (SEQ ID NO:6) prepared in a similar way to thatdescribed in Example 6(d) (46 uM protein; 0.10 ml) was mixed with TCEP(5 mM; 0.0037 ml) and incubated at room temperature for approx. 18 h.0.01 ml of 0.5M ethanolamine was added. 0.03 ml of this 0.11 ml solutionwas mixed with 0.0032 ml of MSWP-3 (see Example 4; 2 mM in 0.1M sodiumphosphate pH 7.0); the solution was incubated for a further 3 h at roomtemperature. SDS PAGE analysis showed the major band in the preparationhad an apparent molecular weight of 23,000, clearly shifted from theoriginal parent molecular weight of 21,000. The purity of the targetprotein was estimated from the SDS PAGE gel to be approx. 80%.

Example 14 Preparation of [SCR1-3]-Cys-S-S-[TCPT-1] (This sequence is aconjugate of SEQ ID NO:6 and the base peptide of SEQ ID NO:20).

[SCR1-3]-Cys protein prepared in a similar way to that described inExample 6(d) (46 uM protein; 0.08 ml) was mixed with TCEP (5 mM; 0.0029ml) and incubated at room temperature for approx. 18 h. 0.008 ml of 0.5Methanolamine was added. 0.04 ml of this 0.088 ml solution was mixed with0.0029 ml of TCPT-1 (see Example 5; 29 mM in DMSO). The TCPT-1 was addedin 6 aliquots over a 2 h period to minimise aggregation. The solutionwas incubated for a further 2 h at room temperature. The finalappearance of the mixture was one of a colloidal suspension andcentrifugation at 2000 g for 1 min showed that the target protein wascompartmentalised in the precipitate. SDS PAGE analysis showed the majorband in the preparation had an apparent molecular weight of about23,000, clearly shifted from the original parent molecular weight of21,000. The purity of the target protein was estimated from the SDS PAGEgel to be approx 80%.

Example 15 Preparation of a Rabbit anti-(human erythrocyle membrane)antibody-[MSWP-1] conjugate (RAEM-MSWP-1) (This sequence is a conjugateof Rabbit anti-human erythrocyle membrane antibody and the base peptideof SEQ ID NO:5).

Rabbit polyclonal and (human erythrocyle membrane) (RAEM) antiserum(Dako, Denmark, 13 mg/ml, 0.25 ml) was diluted to 1.0 ml with 50 mMsodium phosphate 0.1M sodium chloride pH 7.4 (PBS) and treated with 30ml of 100 mM 2-iminothiolane in PBS (freshly dissolved) for 30 min at25° C. These conditions have been shown (R. A. G. Smith & R. Cassels,Fibrinolysis, 2, 189-195, 1988) to introduce an average of 2-3 freethiol groups per molecule of immunoglobulin G.

The product was purified by gel permeation chromatography on a smalldisposable columns of Sephadex G-25 m (PD-10, Pharmacia, Stockholm,Sweden) at 4° C. 2.5 ml of the product (total volume 3.0 ml, theoreticalprotein concentration −6.1 mM) was treated with MSWP-1 (Example 2, 0.125m of 5 mM solution in dimethyl sulphoxide, final conc −240 uM) andincubated at 25° C. for 30 min. The product was gel-filtered on a PD10column as above to give 3.0 ml of a solution −5 uM in protein. This wasstored frozen at −70° C.

Example 16 Preparation of a conjugate of Streptokinase and MSWP-1 (Thissequence is a conjugate of Streptokinase and the base peptide of SEQ IDNO:5).

Streptokinase (SK) stock solution (Behringwerke, Marburg, Germany, 12.8mg/ml, 271 uM, 2.5 ml) was gel filtered using a PD10 column into 3.2 mlof PBS buffer (see Example 15) containing 0.01% w/v Tween 80 [PSTbuffer]. Freshly made up 2-iminothiolane (64 ul of 100 mM) was added andthe mixture incubated at 25° C. for 1 h. The product was gel filtered in2×1.6 ml batches into 2×3.0 ml PST at 4° C. on two PD10 columns. Thissolution was stored in aliquots of 1.5 ml at −75° C.

Titration of the product with Ellman's reagent (0.1 mM in 0.5 ml of 0.1MTriethanolamine, HCl pH 8.0) showed that it contained approximately 0.3mM free thiol groups. This corresponds to an average of 3-3.5 thiolgroups per molecular of SK. The stock thiolated SK solution (2×0.5 ml)was processed by modifying one aliquot with MSWP-1 (32 ml of 5 mM stockin DMSO), incubated 1 h at 25° C. and gel filtered (PD10 column) late3.0 ml PST at 4° C. A control aliquot was processed in parallel withoutexposure to MSWP-1. Both products contained −0.8 mg/ml protein based onas extinction coefficient of 0.76 (mg/ml)⁻¹ at 820 nm for SK and werestored at −75° C.

Example 17 Reversible linkage of MSWP-1 to the active center of HumanTissue-type Plasminogen Activator (This sequence is a conjugate of SEQID NO:51 and the base peptide of SEQ ID NO:5).

The thiol-reactive acyl-enzyme4-N-(2-N-(3-[2-pyridyldithio]-ethylcarbonyl)aminoethyl]aminobenzoyl-[Ser-478]human tissue-type plasminogen activator [PDAED->tPA] was prepared by themethod of Smith and Cassels (Fibrinolysis, 2, 189-195, 1988). Tissueplasminogen activator (Actilyse, Boehringer Ingelheim, Germany, approx 2mg) was dissolved in the PST buffer of Example 16 (1.0 ml) and treatedwith 25 ul of a 20 mM solution of 4′-amidinophenyl4-N-[2-N-(3-[2-pyridyldithio]-ethylcarbonyl)aminoethyl]aminobenzonatehydrochloride (S. B. Kalindjian & R. A. G. Smith, Biochem. J. 248,409-413, 1987) in dimethylsulphoxide. The mixture was incubated for 1 hat 25° C. and stored frozen at −80° C. It was reduced by addition ofdithiothreitol (5 ul of 0.5M in water) for 30 min at 0° C. followed bybuffer-exchange into PST buffer (3.0 ml) as described in Example 16. Theproduct was divided immediately into a retained sample (0.6 ml) and areaction sample (2.4 ml) which was mixed with MSWP-1 (Example 2, 100 ulof a 5 mM solution in dimethylsulphoxide) and incubated for 90 min onice. The product was buffer-exchanged as above into 3.2 ml PST andstored in aliquots at −196° C.

Example 18 Expression and purification of [SCR1-3]-Cys (SEQ ID 6) from aformentation run.

(a) Formentation of E. coli harbouring the plasmid pDB1030

A frozen stock of E. coli harboring the plasmid pDB1030 was initiallyprepared by plating the culture out onto LB agar plus ampicillin at 100μg/ml. 1 ml aliquots were preserved in a 10% glycerol/PBScryopresservative and stored under liquid nitrogen. A 1 ml vial wasthawed and was used to inoculate 100 ml LB^(Amp100) primary seed medium(Difco Bactotryptose, 10 gl⁻¹; Difco yeast extract, 5 gl⁻¹; sodiumchloride, 5 gl⁻¹; pH prosterilisation 7.4) in a 500 ml flask. Theprimary seed stage was incubated at 37° C. for 3 hours before transferto the second seed stage, also 100 ml LB^(Amp100) per 500 ml flask usinga 1% inocultum. Following incubation as above for a further 4 hours a 1%inoculum was transferred to the tertiary seed stage, 10 litersLB^(Amp100) in a 15 liter Biolafite fermenter. The 10 liters tertiaryseed medium was sterilised in situ for 45 minutes at 121° C. beforeinoculation. Following incubation for 14.5 hours, the tertiary seed wastransferred to the final stage fermenter as a 6% inoculum. Incubationconditions for the seed stage were as follows: airflow at 101 min⁻¹ (1.0vvm), temperature 37° C., agitation at 400 rpm (1.9 ms⁻¹) andoverpressure 0.2 bar. 300 liters Tryptone phosphate medium^(Amp100)(Difco Bactotryptone, 20 gl⁻¹; Difco yeast extract, 15 gl⁻¹; sodiumchloride, 8 gl⁻¹; disodium hydrogen orthophosphate, 2 gl⁻¹; potassiumdihydrogen orthophosphate, 1 gl⁻¹; Dow Corning 1520 antifoam, 0.1 gl⁻¹;pH pre-sterilisation 7.4) was sterilised in situ for 30 minutes at 121°C. in a 450L Bioengineering fermenter. The fermenter was inoculated with20 liter inoculum from the tertiary and stage and incubated under thefollowing conditions:airflow 450L min⁻⁴ (1.5 vvm), temperature 37° C.,agitation 230 rpm (1.5 ma⁻¹) and overpressure 0.5 bar. After anOD_(530 mm) of 3.5 was obtained, 1 mM IPTG was added. Harvest followedafter continued incubation for 2 hours. A cell slurry was recoveredafter primary centrifugation through a Westfalia CSA19 (two discharges).The calls were further spun at 4700 rpm (7000 g) for 30 minutes in aSorvall RC3B centrifuge. The total cell yield (wet weight) was 2.98 Kgand was stored at −80 degrees C. in approx. 600 g lots.

(b) Isolation of inclusion bodies and purification of [SCR1-3]-Cys

Inclusion bodies from 100 g (wet weight) cell pellet were isolated andsolubilised essentially as described in Example 6. The purification oftarget protein from resolubilised inclusion bodies was also as describedin Example 6 with some modifications. The major ones were:

1. The use of Macroprep High S (Biorad) instead of S-Sepharose. 200 g ofmatrix was used for 100 g of cell pellet that had been sonicated. 1.4 gof approx. 60% pure target protein was produced in the solubilised andpartially purified fractineon bodies.

2. Refolding of a 100 mg sample of the partially purified protein wascarried out by diluting the fully denatured protein (2 mg/ml) 100-foldin cold 60 mM ethanolamine/1 mM EDTA, followed by addition of theglutathione redox couple.

The product of the above process was capable of being modified withMSWP-1 (Example 2) in a way similar to that described in Example 1.

Example 19 Expression and isolation of[SCR1-3(delN195-K196)]TNANKSLSSISCQT (SEQ ID NO:14)

(a) Construction of plasmid pBC04-29 encoding[SCR1-3(delN195-K196)TANANKSLSSISCQT (SEQ ID NO:14)

Plasmid pBC04-29 was constructed from plasmid pDB1013-5 encoding SCR1-3of LHR-A of CR1 (patent application WO 94/00571) by QuickChange sitedirected mutagenesis (Stratagene) according to the manufactureprotocols. Two complementary oligonucleotides (SEQ ID No 15 and SEQ IDNo 16) were used to generate a novel restriction site (silent) atG186/P187 and a C terminal cysteine. In the event the reaction produceda frame-shift mutation at position N195. In the resulting sequence the Cterminal amino acids N195 and K196 are replaced with a 14 amino acidpeptide TANANKSLSSISCQT (residues 196-209 of SEQ ID NO:14).Fortuitously, this sequence contains an internal cysteine close to the Cterminus, preceeded by a spacer sequence of 11 amino acids.

(b) Expression of plasmid pBC04-29 encoding[SCR1-3(delN195-K196)TANANKSLSSISCQT (SEQ ID NO:14) in E. coli

pBC04-29 was transformed into competent E. coli BL21(DE3)pLys-S andresultant colonies were isolated and checked for plasmid content. Asingle colony was inoculated into 10 ml LB medium (10 g/L bactotryptone,5 g/L yeast extract, 10 g/L NaCl) containing 50 ug/ml ampicillin. Theculture was grown for 6-18 hours at 37° C., 230 r.p.m. before being usedto inoculate 1 liter of the same medium containing 50 ug/ml ampicillinat a dilution of 1 in 100 in 4 L erlenmeyer flasks. Cells were grows toan OD of 0.8-1.0 at A₆₀₉ nm. IPTG (isopropyl B-D galactopyranoside) wasadded to a final concentration of 1 mM and cells allowed to continuegrowth for a further 3-4 hours or overnight before harvesting bycentrifugation at 8000 g/10 min. Pellet from 1L of culture was stored at−80° C.

(c) Isolation and purification of [SCR1-3(delN195-K196)TANANKSLSSISCQT(SEQ ID NO:14)

The methods are essentially those detailed in Dodd L et al. (1995)Protein Expression and Purification 6 727-736, subsequently modified asdescribed in Example 18. Briefly, the cell pellet from 1L of culturefrom (b) was resuspended in buffer, sonicated and the inclusion bodiesisolated by centrifugation. The inclusion bodies were resolubilised in100 ml of fully reducing buffer and target protein purified on MacroprepHigh S (30 g wet weight). Product (27 ml at nominal 1.5 mg/ml) thateluted from the column in the 1M NaCl-containing buffer was refolded bydilution into 2.5 L cold 60 mM ethanolamine/1 mM EDTA, with theglutathione redox agents added at 1 h. After 3 d at 4 degrees C. thesolution was ultrafiltered using a YM10 membrane and the retentate wastreated with ammonium sulphate, centrifugated and the supernatantpurified on Butyl Toyopearl 650M (bed volume 53 ml). A single A280 peakwas eluted by the decreasing ammonium sulphate gradient. SDS PAGE undernon-reducing conditions followed by protein staining revealed a majorpolypeptide with an apparent molecular weight of 22,000, believed to bethe target protein. One of the contaminating polypeptides had asapparent weight of about 40,000, which was identified as the dimer ofthe monomeric form of the target by comparison with adjacent markers of[SCR1-3]-Cys. The product had as estimated protein concentration of 30nM.

Example 20 Preparation of[SCR1-3(deln195-K196)]TNANKSLSSISCQT-(-S-S-[MSWP-1]) (This sequence is aconjugate of SEQ ID NO:14 and the base peptide of SEQ ID NO: 5).

[SCR1-3(delN195-K196)TANANKSLSSISCQT (SEQ ID NO: 14) prepared asdescribed in Example 19 (approx. 30 uM protein; 0.1 ml) was mixed withTCEP (5 nM in 50 mM Hepes pH 4.5; 0.0072 ml) and incubated at roomtemperature (22 degrees C.) for 15 h. 0.05 ml of this solution was mixedwith 0.005 ml of 0.5M ethanolamine and 0.003 ml of 7 mM MSWP-1 (seeExample 2); the solution was incubated for a further 4 h at roomtemperature. SDS PAGE analysis showed a major band in the preparationhad an apparent molecular weight of 25 000, clearly shifted from theoriginal parent molecular weight of 23 000.

Example 21 Preparation of [SCR1-3]DGPSEILRGDFSSC (SEQ ID No. 23)

(a) Construction of plasmid pBC04-31 encoding [SCR1-3]DGPSEILRGDFSSC(SEQ ID NO:23)

Plasmid pBC04-31 was constructed using plasmid pBC04-29 (described inExample 19) and a synthetic oligonucleotide pair (SEQ ID No. 25 and SEQID No. 26). pBC04-29 was digested with the restriction enzymes HindIIIand Apal and the large fragment (2170 bp) isolated. The twooligonucleotides were annealed by warming to >90° C. and slowly coolingto room temperature and ligated with the DNA fragment. The ligated DNAwas transformed into competent E. coli XLI-Blue. Colonies were analysedfor plasmids in which the oligonucleotides had been inserted by lookingfor the presence of a novel Aval site at position 2733. On digestionwith Aval pBC04-31 yielded fragments of 2311 and 495 bp. DNA frompositive clones was used to transform the expression strains. Theoligonucleotides inserted added the peptide sequence DGPSEILRGOFSSC(residues 198-211 of SEQ ID NO:23) to the C terminus of SCR1-3 and alsorepaired the frame-shift error seen in pBC04-29.

(b) Expression, isolation and purification of [SCR1-3]GDPSEILRGOFSSC(SEQ ID NO: 23)

Expression, isolation and purification of [SCR1-3]DGPSEILRGDFSSC (SEQ IDNO: 23) is carried out using pBC04-31 by procedures generally describedin Example 6.

Example 22 Preparation of [SCR1-3]DGPSEILRGOFSSC-(-S-S-[MSWP-1]) (Thissequence is a conjugate of SEQ ID NO:23 and the base peptide of SEQ IDNO:5).

(SCR1-3)]DGPSEILRGDFSSC (SEQ ID NO:23) protein prepared in a similar wayto that described in Example 21 is reacted with MSWP-1 as described inExample 8 to give the title compound.

Example 23 Preparation of [SCR1-3]AAPSVIGFRILLLKVAGC (SEQ ID No. 33)

(a) Construction of plasmid pBC04-34 encoding [SCR1-3]AAPSIGFRILLKVAGC(SEQ ID NO:33)

Plasmid pBC04-34 was constructed using plasmid pBC04-29 (described inExample 19) and a synthetic oligonucleotide pair (SEQ ID No. 34 and SEQID No. 35). pBC04-29 was digested with the restriction enzymes HindIIIand Apal and the large fragment (2170 bp) isolated. The twooligonucleotides were annealed by warming to >90° C. and slowly coolingto room temperature and were ligated with the DNA fragment. The ligatedDNA was transformed into competent E. coli XLI-Blue. Colonies wereanalysed for plasmids in which the oligonucleotides had been inserted bylooking for an increase in size of the Ndel/HindIII fragment by 50 basepairs. The presence of the cysteine codon was determined by the presenceof a Ddel site at position 2781. pBC04-34 digested with Ddel yieldeddiagnostic bands of 481 and 109 bp. DNA from positive clones was used totransform the expression strains (see next section). Theoligonucleotides inserted added the peptide sequence AAPSVIGFRILLLKVAGC(SEQ ID NO: 43) to the C terminus of SCR1-3 and also repaired theframe-shift error seen in pBC04-29.

(b) Expression, isolation and purification of [SCR1-3]AAPSVIGFRILLLKVAGC(SEQ ID NO:33)

Expression, isolation and purification of [SCR1-3]AAPSVIGFRILLLKVAGC(SEQ ID NO:33) is carried out using pBC04-34 by procedures generallydescribed in Example 6.

Example 24 Preparation of [SCR1-3]AAPSVIGFRILLLKVAGC-(-S-S-[MSWP-1])(This sequence is a conjugate of SEQ ID NO:33 and the base peptide ofSEQ ID NO:5).

[SCR1-3]AAPSVIGFRILLLKVAGC (SEQ ID NO:33) protein prepared in a similarway to that described in Example 23 is reacted with MSWP-1 as describedin Example 8.

Biological Activity

(I) Anti-complement Activity Measured by the Classical Pathway-mediatedHaemalysis of Sheep Erythrocyle

(i) Functional activity of complement inhibition was assessed bymeasuring the inhibition of complement-mediated lysis of sheeperythrocytes sensitised with rabbit antibodies (Diamedix Corporation,Miami, USA). The assay is designed to be specific for the classicalpathway of complement activates. Human serum diluted 1:500 or 1:400(final concentration in assay mixture) in 0.1M Hepes/0.15M NaCl/0.1%gelatin pH 7.4 was used as a source of complement. The serum wasprepared from a pool of volunteers essentially as described in Dacia &Lewis, 1975. Briefly, blood was warmed to 37° C. for 5 minutes, the clotremoved and the remaining serum clarified by centrifugation. The serumfraction was split into small aliquots and stored at −196° C. or −80° c.Aliquots were thawed as required and diluted in the Hepes bufferimmediately before use.

Inhibition of complement-mediated lysis of sensitised sheep erythrocyteswas measured using a standard haermolytic assay using a v-bottommicrotitre plate format as follows:

50 μl of a range of concentration of inhibitor diluted in Hepes bufferwere mixed with 50 μl of the diluted serum and 100 μl of sensitisedsheep erythrocytes and then incubated for 1 hour at 37° C. Samples werespun at 1600 rpm at ambient temperature for 3 minutes beforetransferring 150 μl of supernatant to a flat bottom microtitre plate anddetermining the absorption at 405 or 410 nm. Maximum lysis (Amax) wasdetermined by incubating serum with erythrocytes in the absence of anyinhibitor. Background lysis (Ao) was determined by incubatingerythrocytes in the absence of any serum or inhibitor. Inhibition wasexpressed as a fraction of the total cell lysis such that IH50represents the concentration of inhibitor required to give 50%inhibition of lysis.

% inhibition=1-[(A-Ao)/(Amax-Ao)]

Results Compound IH50 WO94/00571 0.2-0.3 ug/ml [10-15 nM] (1) SEQ ID NO27 Example 6* 0.65 ug/ml [30 nM] (mean of two) (2) Example 7* 0.3-1.0ug/ml [15-50 nM] (n = 3) Example 8a 0.014 ug/ml [0.6 nM] ) Example 8b<0.001 ug/ml [<0.04 nM] ) (3) Example 8c 0.001 ug/ml [0.043 nM] )Example 8d⁺ [0.06 nM] ) Example 10a 0.02 ug/ml [0.8 nM] Example 10b−0.01 ug/ml [−0.4 nM] Example 12 −0.0016 ug/ml [0.07 nM] Example 13−0.009 ug/ml [0.4 nM] Example 14 −1.1 ug/ml [50 nM] Example 19 [4 nM]*As 2-mercaptoethanol/g lutathione derivatives ⁺Assay of the twosolutions and the original pre-lyophilisation solution from Example 8d.Other IH₅₀ values generated for similar batches include: (1) 15 nM (2) 8nM, 5 nM, 8 nM, 4 nM (3) 0.3 nM, 0.2 nM, 0.07 nM, 0.06 nM, 0.2 nM, 0.4nM, 0.5 nM, 0.6 nM.

The above data show that:

1. The complement inhibitory activities of the ‘base’ protein (SCR1-3 ofhuman complement receptor 1 of WO94/00571) and its derivatives witheither an additional C-terminal cysteine (SCR1-3/cys, Example 6) or asingle cationic ‘switch’ sequence (SCR1-3/switch, Example 7) aresimilar.

2. However, incorporation of two membrane binding elements (electronicswitch and myristoyl) by addition of MSWPs-1, 2 or 3 (which contain bothelements) to SCR1-3/cys or three membrane binding elements by additionof the MSWP-1 to the SCR1-3/switch construct results in products whichare significantly more potent (−20-200x) than the base or singlemembrane binding element proteins. The use of TCTP-1 which is targetedto membrane elements found in CD3-positive cells and not to erythrocytemembranes gave a conjugate of similar potency is SCR1-3 derivatives withno or single membrane addresses. Thus, the increases is potency in anassay which depends on an erythrocyte membrane event (cytolysis by themembrane attack complex of complement) can be attributed to membranetargeting of the cytolysis inhibitor proteins by the combination of twomembrane binding elements.

(II) Assay of Anti-complement Activity in the Classical PathwayHaermolytic Assay: activity in the sera of domestic pig, guinea pig, ratand marmoset.

The activity of [SCR1-3]-Cys-S-S-[MSWP-1] was examined in the classicalpathway haermolytic assay using the sera of pig, guinea pig, rat ormarmoset. The methodology was essentially as described in (I) with minormodifications, for example small changes to the concentration of serumused. [SCR1-3]-Cys-S-S-[MSWP-1] was prepared essentially as described inExample 8c. The IH50 values for the different sera were: pig, 0.2 nM;guinea pig, 0.3 nM; rat, 0.4 nM; marmoset, 0.2 nM. These results showthat [SCR1-3]-Cys-S-S-[MSWP-1] is capable of inhibiting classicalpathway complement inhibition in the sera of a variety of animalspecies.

(II) Anti-complement Activity Measured by Alternative Pathway-mediatedHaemolysis of Guinea Pig Erythrocyle

Functional activity of complement inhibitors was assessed by measuringthe inhibition of complement mediated lysis of guinea pig erythrocytesessentially as described by Scesney, S. M. et al (1996) J. Immunol, 261729-1735. The assay is designed to be specific for the alternativepathway of complement activation. Human serum prepared from a pool ofvolunteers essentially as described in Dacie & Lewis, 1975 was used asthe source of complement. Briefly, blood was warmed to 37° C. for 5minutes, the clot removed and the remaining serum clarified bycentrifugation. The serum fraction was split into small aliquots andstored at −196° C. or −80° C. Aliquots were thawed as required anddiluted in 0.1M Hepes/0.15M NaCl/0.1% gelatin/8 mM EGTA/5 mM MgCl₂ pH7.4 (buffer A) immediately before use. Guinea pig erythrocytes wereprepared from guinea pig whole blood collected into EDTA-coated tubes asfollows. The blood was span at 1600 rpm for 5 min and the erythrocytepellet washed 3 times with 0.1M Hepes/0.15M NaCl/0.1% gelatin pH 7.4until the supernatant of the spin was essentially colourless. Theerythrocytes were finally resuspended to the original volume of bloodused and were stored at +4 degrees C. they were used within 2 weeks.

50 μl of a range of concentrations of inhibitor diluted in buffer A is av-bottom microtitre plate were mixed with, first, 100 μl of serum thathad been diluted 1:3 and second, 50 μl of guinea pig erythrocytes(diluted 1:49 in buffer A) and incubated for 1 hour at 37° C. The planwas span at 1600 rpm for 3 minutes before transferring 150 μl of eachsupernatant to a flat bottom microtitre plate and determining theabsorption at 405 nm, which reflects the amount of lysis in each testsolution. Maximum lysis (Amax) was determined by incubating serum witherythrocytes in the absence of any inhibitor. Background lysis (Ao) wasdetermined by incubating erythrocytes in the absence of any serum orinhibitor. The final dilution of serum used in the assay did absorb at405 cm but the level of absorbance (approx 10% of Amax) was consideredto have a neglible affect on the overall assay results and it wasignored in the calculations. Inhibition was expressed as a fraction ofthe total cell lysis such that IH50 represents the concentration ofinhibitor required to give 50% inhibition of lysis.

% inhibition=1-[(A-Ao)/(Amax-Ao)]

Results

Two aliquots (one lyophilised and resolubilised in a neutral buffer, theother not lyophilised) of a single batch of o([SCR1-3]-Cys-S-S-[MSWF-1]prepared in a similar way to that described in Example 8 (c) were testedin the haermolytic assay. The IH50 values for the compounds were:

[SCR1-3]-Cys-S-S-[MSWP-1] (not lyoph) 310 nM [SCR1-3]-Cys-S-S-[MSWP-1](lyoph) 480 nM

The result shows that [SCR1-3]-Cys-S-S-[MSWP-1] exhibited activityagainst the alternative pathway of the complement system and thatlyophilisation and subsequent resolubilisation of the protein had noaffect (within experimental error) on the biological activity of theprotein.

(III) Plasminogen Activator Assay

(j) SK-related molecules from Example 16 were assayed using aplasminogen activation assay. A solution of purified humanLys_(n)-Plasminogen (1 uM in PST buffer containing 25% v/v glycerol[PSTG buffer], 0.5 ml) was incubated with thiolated SK (finalconcentration 0.1 to 1.07 nM) for 1 h at 25° C. An aliquot of thismixture (10 ul) was incubated with 1.0 mM of the plasmin substrateS-2251 (H-D-Val-Leu-Lys-p-nitroanilide, KabiVitrum, Stockholm, Sweden)in 0.1M Triethanolamine HCl pH 8.0 (0.5 ml) at 25° C. The release ofp-nitroaniline was monitored continuously at 405 nm. Under theseconditions, one substrate unit (SU) of plasmin activity is defined asthe amount of enzyme giving an increase in optical density at 405 nm of0.001 min⁻¹. Under these conditions thiolated SK (1 nM), generatedplasmin at a nearly linear of 4225 SU/ml.

SK-MSWP-1 conjugate was diluted 1:100 in PSTG buffer and 5-50 mlaliquots tested in the plasminogen activation assay. The stockpreparation was found to contain approximately 2.9 uM functional SK.

(II) The potential activity of the acyl-enzyme preparations of Example17 was estimated by dilution 25-50 fold into PST buffer and incubationfor 2 h at 37° C., followed by assay using 2 mM (S-2288(H-D-Ila-Pro-Arg-p-nitroanilide 2HCl) under the same conditions used in(i) above. Under these conditions, the potential activity of the reducedPDAEB->tPA preparation was 2760 SU/ml and that of the MSWP/1/PDAEB->tPAconjugate 535 SU/ml.

(IV) Erythrocyte Binding Assays

(i) Erythrocyte aggregation test for modified and unmodified Rabbitanti-(human erythrocyte membrane) antibody.

Human pooled erythrocytes (Ortho A2, Raritan, N.J. 3% v/v, 50 ml) wereadded to microtitre plates wells and either unmodified rabbitanti-(human erythrocyte membrane) antibody [RAEM] or RAEM-MSWP1conjugate from Example 15 added at concentrations expressed relative toundiluted stock RAEM. Cells agitated at −100 rpm for 40 min at 25° C. 5ul was removed from each well and examined by light microscopy at ×20magnification. A visual scoring scale was used as follows:

Results Control (n = 6) − RAEM-MSWP1 1/3900 +/− RAEM 1/1100 − RAEM-MSWP11/1000 +/− RAEM 1/600 − RAEM 1/350 +/− RAEM-MSWP1 1/357 +++ RAEM 1/50 ++RAEM-MSWP1 1/62 +++ − No clumping, cells moving freely relative to eachother. + Small clumps (<10 cells) ++ Larger clumps (100 plus cells) +++Very large visible aggregates

Conclusion

The antibody preparation modified to contain a membrane-binding unit wasmore effective at inducing aggregation of cells because binding to thecell membrane through MSWP1 allowed a higher effective concentration ofbridging antibody on the membrane surface.

(II) Binding of 125-Iodine-[SCR1-3]-Cys-S-S-[MSWP-1] to HumanErythrocytes

[SCR1-3]-Cys-S-S-[MSWP-1] (2 mg/ml in PBS; 0.25 ml) was mixed with 0.5mCl of 125-Iodine (Amersham) in the presence of 9 moles potassium iodidefollowing the Iodogen procedure and reagents (Pierce and Warriner (UK)Ltd.). The labelling was allowed to proceed for 20 min at roomtemperature, the reaction was quenched with 0.1 ml of 1M potassiumiodide and the solution buffer-exchanged into PS/0.1% albumin. Citratedblood collected from a healthy volunteer was used as a source of humanerythrocytes. Blood (0.2 ml) was mixed with 10 microlitres ofappropriately diluted 125-Iodine-[SCR1-3]-Cys-S-S-[MSWP-1] (finalconcentration 700 pM) and incubated for 30 min at 37 degrees C. Theerythrocytes were then isolated by three repeat washings inPSB/centrifugation steps and samples counted in a Wallac 1470 Wizardgamma counter. The results were as follows:

cpan 1st wash 3 600 000 1st pellet 140 000 2nd wash 52 000 3rd wash 6500 final pellet 26 000

Using values of 5×10⁹ erythrocytes per ml of blood and and a specificradioactivity of 2.7×10⁷ cpm/nmole for the [SCR1-3]-Cys-S-S-[MSWP-1] itwas calculated that about 600 molecules of [SCR1-3]-Cys-S-S-[MSWF-1]bound per cell (the values for ‘final pellet’).

(III) Binding of Fluoruresin-labelled-[SCR1-3]-Cys-S-S-[MSWF-1] to HumanErythrocytes

[SCR1-3]-cys (prepared in a similar way to that described in Example 18)(45 uM, 1.0mg/ml in 0.1M sodium phosphate, approx. 0.2M ammoniumsulphate pH 7.0) was partially reduced by incubation at 25° C. for 18 hby the addition of a 4-molar excess of Tris(2-carboxyethyl)phosphine(TCEP; Pierce & Warriner (UK) Ltd.). The solution was buffer exchangedinto 50 mM Hepes pH7.0; post buffer exchange the protein concentrationwas 22 uM. The partially reduced [SCR1-3]-cys was incubated with a4-fold molar excess of 6-(fluorescein-5-carboxamide)hexanoic acid,succinimidyl ester (Molecular Probes Inc. USA) and incubated for 1 h at4° C. the excess fluorescent label was removed by buffer exchange of theprotein solution into 50 mM Hepes pH7.0.Fluorescein-[SCR1-3]-cys-S-S-[MSWP-1] was synthesized by adding MSWP-1(Example 2) to give a five-fold molar excess over fluorescein labelledprotein and was incubated for 4 h at 25° C. The solution was bufferedexchanged into PBS and this solution was used for the microscopystudies.

[SCR1-3], 10 mg/ml in 50 mM f rmic acid, was mixed in a 1:10 ratio with50 mM NaHCO₃ pH8.5; the pH of the solution was adjusted with NaOH topH9.5. The fluorescein was extracted from Celite-fluoresceinisothiocyanate (Celite:fluorescein; 1:10, Sigma) by DMSO in a 1:4 (w/v)ratio. The fluorescein-DMSO solution was added to the protein solutionin a 1:14 ratio and incubated for 1 h at RT. Excess label was removed bygel filteration into PBS containing 0.01% Tween-80 and this solution wasused for microscopy studies.

Citrated blood was collected from a healthy volunteer and theerythrocytes isolated, washed in PBS and diluted 250-fold compared tothe original blood volume. 0.05 ml of erythrocytes were incubated with 2nM fluorescein-[SCR1-3]-cys-S-S-[MSWP-1] or 2 nM fluorescein-[SCR1-3]and incubated for 30 min at 37° C. An eight microliter sample of eachincubation was mounted on a slide and viewed on an inverted confocalmicroscope (Biorad). The cells incubated with fluorescein-[SCR1-3]showed no specific staining whereas with those incubated withfluorescein-[SCR1-3]-cys-S-S-[MSWP-1] staining appeared diffusely overthe cell surface and also intensely stained patches were visible on thecell membrane. No labelling was seen intracellularly.

(iv) Binding of MSWP-1/PDAEB→tPA to human erythrocytes

Human trypsinized and glutaraldehyde-treated red blood cells (1.0 ml ofa 4% suspension) was pelleted by low-speed centrifugation andresuspended is a total volume of 0.5 ml PST containing either noaddition or approximately 270 SU of either reduced PDEAB→tPA orMSWP-1/PDAEB→tPA conjugate of Example 17. The mixtures were incubated bygentle rolling for 5 min at 23° C. and then the cells were pelleted bycentrifugation followed by two washes with 1.0 ml PST buffer. Finally,the cells were suspended in 0.5 ml PST and incubated at 37° C. Samplesof the supernatant (100 ul) were removed after pelleting. Assay usingS-2288 (as above) showed that after 2 h, approximately 7% of the appliedt-PA activity was present in the supernatant of cells exposed toMSWP-1/PDAEB→tPA whereas only −2.8% was present in the supernatant ofcells exposed to reduced PDAEB→tPA alone. No t-PA amidolytic activitywas detected in controls.

This experiment suggests that reversible linkage of the active site oft-PA to MSWP-1 increases the tendency of this enzyme to bind to redblood cells.

(v) Localisation fSK-MSWP-1 conjugate on the surface of humanerythrocytes

A stabilised preparation of human erythrocytes (trypsinized,glutaraldehyde-treated, Sigma, Gillingham, UK, 4% v/v, 0.4 ml) waspelleted by centrifugation (−2000 g/2 min) and resuspended in 0.4 ml PSTbuffer with either 0.1 uM thiolated SK or 0.1 uM SK-MSWP-1 from Example16.

The suspensions were incubated for 30 min at 37° C. and then washed bytwo cycles of centrifugation and resuspension in PST buffer. Finally,they were resuspended in PSTG buffer (0.4 ml) containing 1 uMplasminogen and incubated and assayed for plasmin as described above.

The control thiolated-SK generated plasmin at a rate of 522 SU/ml, whilethe SK-MSWP-1 conjugate produced 6184 SU/ml. The latter activitycorresponds to around 2100 thiolated SK molecules/cell.

(vi) Binding of [SCR1-3]-Cys-S-S-[MSWP-1] to human erythrocyte membranes

4×2.0 ml of trypsinized, glutaraldehyde-treated human erythrocytes(Sigma, R0127) were centrifuged for 2 min at about 3000 rpm. Thesupernatants were discarded and the cells resuspended inphosphate/saline/Tween (0.01%) (PST) (1 ml per tube) and[SCR1-3]-Cys-S-S-[MSWP-1] of Example 8 was added to a finalconcentration of 20 ug/ml to three of the tubes. The mixtures were thenincubated at 37 degrees C. for 30 min., then washed five times by repeatcentrifugation and washing in PST. The cells were finally suspended in 1ml PST and were held at 4 degrees C.

The ability of these cells to inhibit complement-mediated lysis of sheeperythrocytes was measured using the standard classical pathwaycomplement inhibition assay described in (I) above. The humanerythrocytes were added to the assay at four different dilutions,followed by the human serum and then the sheep red blood cells andincubation at 37 degrees C. as usual. The % inhibition data are shownbelow.

human erythrocytes, human erythrocytes, treated with Final dilutionuntreated [SCR1-3]-Cys-S-S-[MSWP1] 1/4 22% 62% 1/16 −8% 88% 1/256 5% 74%1/2500 −7% 51%

Thus the percentage inhibition for the [SCR1-3]-Cys-S-S-[MSWP-1]-treatedcells at maximum dilution was significantly higher than the untreatedcells at ¼ dilution. The [SCR1-3]-Cys-S-S-[MSWP-1]-treated cells,therefore, contained at least 500-fold more complement inhibitoryactivity than the untreated cells, even though the cells had been washedextensively to remove any non-bound [SCR1-3]-Cys-S-S-[MSWP-1].

The following methods and examples further illustrate aspects of theinvention.

Introduction of DNA into E. coli

Plasmids were transformed into E. coli XL1-Blue (Stratagene),HMS174(DE3) (Novagen, UK) or UT5600(DE3) (see below) that had been madecompetent using calcium chloride as described in Sambrook et al,(op.cit.). UT5600 was purchased from New England Biolabs (#801-I) andwas converted to a DE3 lysogen. UT5600 was isolated as a mutant of K12strain RW193 (itself derived from AB1515) which was insensitive tocolicin-B (McIntosh et al. (1979) J.Bact. 137 p653). It was notinitially known that ompT had been lost, but further work by the samegroup showed that protein α (now OmpT) was lacking (Earhart et al (1979)FEMS Micro Letts 6 p277). The nature of the mutation was determined tobe a large deletion (Elish et al (1988) J Gen Micro 134 1355).

DNA sequencing

DNA sequencing was contracted out to Lark (Saffron Walden, Essex UK) orMWG (Milton Keynes, UK).

Production of oligonucleotides

Oligonucleotides were purchased from Cruachern (UK) or Genosys-Sigma(Pampisford, Cambridgeshire UK)

Polymerase chain reaction amplification of DNA

Purified DNA or DNA fragments from ligation reactions or DNA fragmentsexcised and purified from agarose gels were amplified by PCR from twoprimers complementary to the 5′ ends of the DNA fragment. Approximately0.1-1 mg of DNA was mixed with commercially available buffers for PCRamplification such as 10 mM Tris pH 8.3 (at 25° C.), 50 mM KCl, 0.1%gelatin; MgCl₂ concentrations were varied from 1.5 mM to 6 mM to find asuitable concentration for each reaction. Oligonucleotide primers wereadded to a final concentration of 2 mM; each dNTP was added to a finalconcentration of 0.2 mM. 1 unit of Taq DNA polymerase was then added tothe reaction mixture (purchased from a commercial source, e.g. Gibco).The final reaction volume varied from 20 ml to 100 ml, which wasoverlayed with mineral oil to prevent evaporation. Thermal cycling wasthen started in a thermal cycler such as the PCR machine from MJResearch. A typical example of conditions used was 94° C. for 5 minute,55° C. for 1 minute, and 72° C. for 2 minutes; however, the optimaltemperatures for cycling can be determined empirically by workersskilled in the art. The DNA fragment was amplified by repeating thistemperature cycle for a number of times, typically 30 times.

pET15b vector for DAF expression

The pET15b expression vector is a T7 promotor based vector availablecommercially through Novagen (Wisconsin, USA). Briefly, the vectorcarries the following features which make it a useful vehicle for theexpression of heterologous proteins in E. coli; a selectable antibioticmarker (β-lactamase) conferring ampicillin resistance, a copy of thelaci gene providing lac repression in strains of E. coli that are laci′,and the T7-lac promoter. The T7-lac promoter combines the T7 RNApolymerase promoter sequences with the laci repressor binding site fromthe E. coli lactose operon. This reduces expression of the cloned genein the absence of the inducer isopropyl β-D thiogalactopyranoside(IPTG). Downstream of the T7 promoter is a multiple cloning site builtinto a region of sequence which codes for a polyhistidine tag sequence.Translation initiates at the methionine codon at position 332-330 of thevector sequence and proceeds counter-clockwise to yield the followingpeptide: MGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 48). The six histidineresidues allow for purification of the fusion protein by metal chelationchromatography, whilst the GLPVR (amino acids 13-17 of SEQ ID NO: 48)motif constitutes a thrombin cleavage site for removal of the peptidefrom the fusion protein after purification. Three restriction enzymesites are provided for the insertion of cloned DNA in-frame with thepolyhistidine leader. These are Ndel (CATATG), Xhol (CTCGAG) and BamHi(GGATCC). Use of the Ndel site to overlap the methionine initiationcodon of the cloned gene removes the possibility of unwanted amino acidsat the N-terminus of the fusion protein. At the 3′ end of the multiplecloning site is the T7 transcriptional terminator.

Colorimetric determination of protein concentration

Protein concentration determination utilised a colorometric methodutilising Coomassie Plus Protein Assay Reagent (Pierce Chemical Company)according to the manufacturer's instruction. The assay used a referencestandard of the protein of Example 6.

Identification of proteins by Western blot

For certain procedures, it is necessary to characterise the expressionof recombinant proteins by an immunological method termed a Westernblot. In this method, proteins to be analysed are separated by SDS-PAGE,transferred to a protein binding membrane such as polyvinylidenedifluoride (PVDF), and then probed with an antibody that is specific forthe target protein. Typically, the binding of the first antibody isdetected by the addition of an enzyme-labelled secondary antibody and anappropriate solution which contains a chromogenic substrate. Oneprocedure for the transfer of proteins to a protein-binding membrane wasas follows. After SDS-PAGE, the proteins on the gel were transferred byelectrotransfer to a protein-binding surface such PVDF. In thisprocedure, two sheets of filter paper (3M, Whatman) soaked in 0.3M Tris,10% (v/v) methanol, pH10.4, were placed on the anode of anelectroblotter (Semi-dry blotter, Biorad). These filter papers were thenoverlayed by a further two sheets of filter paper soaked in 25 mM Tris,10% (v/v) methanol, pH10.4. On top of this stack of filter papers wasplaced a sheet of PVDF membrane which had been pre-wetted in methanoland then soaked in a buffer that comprises 25 mM Tris, 10% (v/v)methanol, pH10.4. The SDS-PAGE gel was then placed on the top of thePVDF membrane, and overlayed with two sheets of filter paper soaked in25 mM Tris, 192 mM 6-amino-n-caproic acid, 10% (v/v) methanol. Thecathode of the electroblotter was then placed on top of the stack offilter papers, gel and membrane, and the proteins transferred by passinga current between the electrodes at 15V for 30 minutes. Subsequent stepsfor the detection of the transferred proteins were described in theNovex WesternBreeze System (Invitrogen). For the detection of humanCD59, a rat anti-CD59 monoclonal antibody YTH53.1 (Davies et al., J.Exp. Med. 170, 637, 1989) was used together with an enzyme-labeledanti-rat secondary antibody. For the identification of His-tagged DAF,an anti his-tag monoclonal antibody was used.

Purification of CD59 from human urine

Urine was collected into 10 mM sodium azide/5 mM benzamidine overapproximately 48 hrs. The urine was then passed through a fluted coarsefilter to remove aggregates. The urine was then concentrated toapproximately 150 mls in a Pellicon concentrator fitted with a membranecassette with a 10 kDa MW cut-off membrane. Insoluble material wasremoved by centrifugation at 10000×g for 30 minutes. The supernatant wasthen applied to a CNBr-activated Sepharose 4B affinity column preparedwith the rat monoclonal anti-CD59 antibody YTH 53.1 (Davies et al. J.Exp. Med. 170, 637, 1989). The column was washed overnight with 1M NaCland bound material eluted with 4M MgCl₂. The protein content of each 1ml fraction eluted from the column was determined by measuringabsorbance at OD280 nm. The fractions containing the most protein werethen pooled and dialysed through a 10 kDa MW cut off membrane into asolution containing 0.9% NaCl, and then dialysed by a similar procedureinto PBS. The dialysed protein was then concentrated using a stirredcell ultrafiltration device (Amicon) fitted with a 10 kDa MW cut-offmembrane. The material may be further purified by gel filtration in 10mM Hepes, 140 mM NaCl, pH7.4, on a Superdex S-75 fast protein liquidchromatography system (Pharmacia) or Sephadex G-75. This method gave ayield of around 7 mg pure protein from 20L urine.

Expression and purification of recombinant soluble CD59 from CHO cells

Soluble CD59 was expressed in a recombinant form from Chinese HamsterOvary cells as follows. Briefly, the polymerase chain reaction was usedto produce a truncated cDNA encoding soluble CD59 from a full lengthcDNA (Davies et al. J. Exp. Med. 170, 637, 1989). A mutation wasintroduced into the cDNA at codon 18 of the mature protein which changedthe Asn codon for Ala. The procedure for this site-directed mutagenesiscan be performed by a number of methods including the Quickchangemutagenesis kit (Stratagene). To introduce the modified gene into theCHO expression plasmid pDR2EF1alpha, the polymerase chain reaction wasused with two oligonucleotides; the first oligonucleotide wascomplementary to the first seven codons at the N-terminus of the matureCD59 protein; and the 3′ oligonucleotides introduces a termination codonimmediately following the codon for Asn-70 of the CD59 cDNA. Theseoligonucleotides were also designed to contain recognition sequences forrestriction enconucleases compatible with the polylinker site of the CHOexpression vector. The DNA fragment resulting from the PCR amplificationwas ligated into a CHO expression vector and this plasmid transfectedwith calcium phosphate into CHO cells. Cells that had become stabilytransfected were selected from untransfected cells by growth in mediumthat contained the antibiotic hygromycin. Individual transformants werepicked and for each clone the expression of CD59 was analysed by ELISA.The highest expressing clone was chosen for large-scale production ofCD59 using a variety of techniques including the use of cell factories(Nunc).

To purify the CD59, the culture medium was precleared by centrifugationat 10000×g for 30 minutes. The soluble CD59 was then purified using animmunoaffinity column containing the monoclonal antibody YTH53.1 (Davieset al. J. Exp. Med. 170, 637, 1989), as described above. The protein wasthen stored in PBS at concentrations of up to 5 mg/mL at −70° C.

Preparation of C56 euglobulin

C56 euglobulin was an essential reagent that was used for theC5b6-initiated reactive lysis of erythrocytes. C56 euglobulin can begenerated in and purifed from some acute-phase sera from post-traumaindividuals (such as sports injuries, surgery or childbirth). Blood wasdrawn from donors in the acute phase of inflammation and allowed to clotat room temperature. To each 10 mls of serum, 0.5 mls of yeastsuspension was added and the mixture incubated overnight on a rotator atroom temperature. The serum was centrifuged to remove the yeast anddialysed against 0.02M Na/K phosphate, pH 5.4. The precipitate(containing the C56 euglobulin) was collected by centrifugation andredisolved in 0.01M Na/K phosphate/0.05M NaCl, pH7.0 containing 25% v/vglycerol.

C5b6-initiated reactive lysis of erythrocytes

Guinea pig erythrocytes (TCS Microbiological, UK) were washed twice inPBS and resuspended to 5% by volume in PBS/0.05% CHAPS. 50 ml of thesecells were placed in the wells of a round-bottomed microtiter plate.Samples to be tested were diluted in PBS/0.05% CHAPS and 50 ml of thesetest solutions added to the wells containing the guinea pigerythrocytes. The plate was then incubated at 37° C. for 20 minutes toallow binding of the samples to be erythrocytes. The microtitre plateswere then centrifuged at 1000 rpm for 5 minutes to pellet the cellsusing a benchtop centrifuge. The supernatants were removed and the cellpellets resuspended in 50 ml PBS/10 mM EDTA. To this cell suspension wasadded 10 ml of a C56 euglobulin solution that varied in concentration indifferent experiments from between 1:50 to 1:500 dilution in PBS/10 mMEDTA. This solution was mixed with the cells by placing the microtitreplate on a microtitre plate shaker for 2 minutes. To this solution wasthen added a 90 ml of a dilution of normal human serum (from 1:50 to1:500 in PBS/10 mM EDTA). The solutions were mixed by placing themicrotitre plate on a plate shaker for a further 2 minutes. The platewas then incubated at 37° C. for 30 minutes. To determine the degree ofhaemolysis, the plate was then placed in a benchtop centrifuge and spunat 1800 rpm for 3 minutes. 100 ml of the supernatant was transferred toa clear flat bottomed microtitre plate and the absorbane at 410 nmmeasured spectroscopically. As controls, guinea pig erythrocytes weretreated in an identical manner to the test samples with the followingexceptions. In the first stage of the assay, the control samples wereincubated with 50 ml of PBS/10 mM EDTA for 20 minutes at 37° C. Aftercentrifugation, a spontaneous lysis control was prepared by resuspendingthe cells in 150 ml PBS/10 mM EDTA; by contrast, for the maximum lysiscontrol, the cells were resuspended in 150 ml water.

Brief Overview of Examples 25 to 36

Example 25

Synthesis and characterization of a membrane-targeted derivative ofsoluble human urinary CD59 (APT632).

Example 26

Synthesis and characterization of a membrane-targeted derivative ofhuman recombinant soluble CD59 (APT637).

Example 27

An alternative Method for the production of urinary (APT2047) andrecombinant (APT2059) human CD59 membrane-targeted derivatives usinglinkage through protein carbohydrate.

Example 28

A method for the preparation of recombinant human CD59 with a C-terminalcysteine, expressed in yeast (APT633).

Example 29

A method for the preparation of recombinant human CD59 with a C-terminalcysteine, expressed in E. coli (APT635).

Example 30

A method for the preparation of recombinant human CD59 with a C-terminalcysteine, expressed in baculovirus/insect cells (APT2060).

Example 31

A method for the preparation of recombinant human CD59 with a C-terminalcysteine, expressed in Chinese hamster ovary cells (APT2061).

Example 32

A Method for the conjugation of the membrane-localising agent APT542 toAPT633, APT635, APT2060 or APT2061.

Example 33

Synthesis and characterization of APT2057 (Human DAF short consensusrepeats 2-4).

Example 34

Synthesis and characterization of APT2058 (Human DAF short consensusrepeats 1-4).

Example 35

Synthesis and characterization of APT2160 (APT2058 derivatized withAPT542).

Example 36

Synthesis and characterization of APT2184 (APT2057 derivatized withAPT542).

Example 25 Synthesis and Characterization of Urine-derived CD59 linkedto MSWP-1 (APT632)

APT632 was synthesized in two steps from soluble CD59 isolated fromhuman urine (APT631; SEQ. ID NO: 37) as described in Methods. APT631 inPBS (200 μL of a 1.9 mg/mL solution) was mixed with 2-iminothiolane (2μL of a 100 mM solution) and the mixture incubated at room temperaturefor 30 minutes. The solution was then dialysed into PBS to removeunreacted 2-iminothiolane, and a solution of tris-2-carboxyethylphosphine (4 μL of a 10 mM solution in 10 mM Hepes, pH7.4) added, andthe mixture left overnight at room temperature. To this solution, 10 μLof APT542 (MSWP-1; 21 mM in dimethyl sulphoxide; SEQ. ID NO. 38) wasadded and incubated at room temperature for 2 h. The product APT632 wascharacterized by the appearance of a protein species that migrated atapproximately 21 kDa as analysed by SDS-PAGE. A reactive lysis assay(described in Methods) demonstrated that APT632, at concentrationsgreater than 0.5 nM, protected guinea pig erythrocytes fromcomplement-mediated lysis by a 1:100 dilution of human serum; bycontrast, no significant protection from lysis was observed with theunmodified form (APT631).

Example 26 Synthesis and Characterization of Recombinant CD59 Producedin CHO Cells Linked to MSWP-1 (APT637)

APT637 was synthesized in two steps from soluble human CD59 that isexpressed in a recombinant form from chinese hamster ovary cells(APT634; SEQ ID NO: 39). APT634 in PBS (200 μL of a 300 μM solution) wasmixed with 2-iminothiolane (6 μL of a 10 mM solution) and the mixtureincubated at room temperature for 30 minutes. The solution was thendialysed into PBS to remove unreacted 2-iminothiolane, and a solution oftris-2-carboxyethyl phosphone (4 μL of a 10 mM solution in 10 mM Hepes,pH7.4) added, and the mixture left overnight at room temperature. Tothis solution, 10 μL of APT542 (21 mM in dimethyl sulphoxide) was addedand incubated at room temperature for 2 h. The product APT637 wascharacterized by the appearance of a protein species which migrated atapproximately 10 kDa as analysed by SDS-PAGE as described in methods. Areactive lysis assay (described in Methods) demonstrated that APT637, atconcentrations greater than 0.5 nM, protected guinea pig erythrocytesfrom complement-mediated lysis by a 1:100 dilution of human serum; bycontrast, no significant protection from lysis was observed with theunmodified form (APT634).

Example 27 A Method for the Production of CD59 Derivatives Linked toMSWP-1 via a Carbohydrate Linkage (APT2047 and APT2059)

APT2047 is a conjugate of APT634 (SEQ ID NO: 39) and APT542 (SEQ ID NO:38). and APT2059 is a conjugate of APT631 (SEQ ID NO: 37) and APT542, inwhich the linkage of each pair of compounds is through a modifiedcarbohydrate moiety on the CD59 protein. APT2047 and APT2059 aresynthesized in three steps from APT634 or APT631. The first stepinvolves the reaction of the proteins APT634 or APT631 at aconcentration of 1 mg/ml with 10 mM sodium periodate for 1 h in thedark, in a solution of 0.1M sodium acetate, pH5.5 To this mixture isadded glycerol to a final concentration of 15 mM and the solution placedon ice for 5 minutes. The mixture is then dialysed into 0.1M sodiumacetate, pH5.5 to remove excess sodium periodate and glycerol. In thesecond step, the sodium periodate-treated proteins are reacted with asolution of (4-[4-N-maleimidophenyl]butyric acid hydrazide hydrochloride(MPBH) at a final concentration of 1 mg/ml for 2 h with stirring. Afterthis procedure, unreacted MPBH is removed by dialysis into a solution of0.1M phosphate, pH7.0, 50 mM NaCl. In the third step of the synthesis,the proteins treated with MPBH are reacted with a solution comprising a5-fold molar excess of APT544 to CD59 for 2 h at room temperature togenerate APT2047 and APT2059. The synthesis of these proteins isconfirmed by the appearance of a novel proteinaceous species thatmigrates at approximately 10 kDa or 20 kDa by SDS-PAGE undernon-reducing conditions, respectively. In addition, these proteinsprotect guinea pig erythrocytes from complement-mediated lysis by humanserum at a concentration greater than 0.5 nM.

Example 28 A Method for the Preparation of Recombinant Human CD59 with aC-terminal Cysteine, Expressed in Yeast (APT633)

APT633 is a protein that comprises soluble human CD59 and a C-terminalcysteine residue following position 81 of the mature CD59 protein. Theprotein was expressed in a recombinant form in Pichia pastoris cells.The polymerase chain reaction was used to produce a truncated cDNAencoding soluble CD59 from a full length cDNA (Davies et al. J. Exp.Med. 170, 637, 1989). The 5′ oligonucleotide was complementary to 20bases of the first 7 codons at the N-terminus of the nature CD59protein, and the 3′ oligonucleotide introduced a cysteine codon and atermination codon immediately following the codon for Ser-81 of themature CD59 protein. These oligonucleotides were also designed tocontain recognition sequences for restriction endonucleases XhoI andEcoRI which are compatible with the polylinker site of the vector pUCP1C(a derivative of pUC19 that contains the alpha-factor leader sequenceand multiple cloning site from pPIC9K (Invitrogen). The DNA fragmentresulting from the PCR amplification was then ligated into pUCPIC DNAand transformed into the XL1-Blue strain of E. coli (Stratagene). Thetransfected cells are selected by growth on a petri dish containing LBmedium (Sigma) supplemented with ampicillin at a concentration of 100micrograms/ml (LBAMP). The DNA from single colonies was isolated andsequenced as described in Methods. The DNA that encodes the alpha factorand CD59 was then subcloned into the vector pPIC9K that had beendigested with the restriction endonucleases BamHI and EcoRI. PurifiedDNA from the resulting plasmid was linearised with the restrictionendonuclease PmeI for transformation into P. pastoris strain GS115(Invitrogen) by spheroplasting according to the manufacturer'sinstructions. After preliminary selection for clones that are capable ofgrowth on a minimal RD medium (1M sorbitol, 2% w/v dextrose, 1.34% yeastnitroen base, 4×10⁻⁵% biotin, 0.005% amino acids) lacking histidine.Clones having undergone multiple integration events were then selectedby resistance to the antibiotic geneticin sulphate (G418). Clones thatwere capable of growth in medium containing G418 at a concentration of 2mg/mL were screened for expression of CD59. Individual colonies wereinoculated in 10 mL BMG medium (100 mM potassium phosphate, pH6.0, 13.4mg/mL yeast nitrogen base, 0.4 mg/L biotin, 1% (w/v) glycerol) and grownat 30° C. with shaking until clones reached an optical density of 6 asmeasured spectroscopically at a wavelength of 600 nm. The cultures werethen transferred to BMM medium (100 mM potassium phosphate, pH6.0, 13.4g/L yeast nitrogen base, 0.4 mg/L biotin, 0.5% methanol) and grown for48 h at 30° C. with shaking. Culture supernatants were then analysed bySDS-PAGE and Western blot for the presence of APT633 which was observedas a novel proteinaceous species which migrated at approximately 8000Da.

Example 29 A Method for the Preparation of Recombinant Human CD59 with aC-terminal Cysteine, Expressed in E. coli (APT635; SEQ ID NO: 41)

APT635 is a protein that comprises soluble human CD59 and a C-terminalcysteine residue following codon 81 of the mature CD59 protein (SEQ IDNO: 41). The protein is expressed in a recombinant form in E. colicells. The polymerase chain reaction was used to produce a truncatedcDNA encoding soluble CD59 from a full length cDNA (Davies et al. J.Exp. Med. 170, 637, 1989). The 5′ oligonucleotide was complementary to20 bases of the first 7 codons at the N-terminus of the mature CD59protein, and the 3′ oligonucleotide introduced a cysteine codon and atermination codon immediately following the codon for Ser-81 of themature CD59 protein. These oligonucleotides were also designed tocontain recognition sequences for restriction endonucleases compatiblewith the polylinker site of pBROC413 (described in WO 94/00571). The DNAfragment resulting from the PCR amplification was then ligated intopBROC413 DNA and transformed into the UT5600(DE3) strain of E. coli(described in Methods). The transfected cells are selected by growth ona petri dish containing LB medium (Sigma) supplemented with ampicillinat a concentration of 100 micrograms/ml (LBAMP). The DNA from singlecolonies was isolated and sequenced as described in Methods. A singlecolony representing UT5600(DE3) cells transfected by DNA encoding APT635was then grown with shaking overnight at 37° C. in LBAMP. This overnightculture was then diluted 1:100 in LBAMP medium and grown with shaking at37° C. until the culture reached an optical density of 1.0 as determinedby absorbance at a wavelength of 600 nm. To this culture was added asolution of isopropyl beta-D-thiogalactopyranoside to a finalconcentration of 1 mM. The culture was then grown for a further 3 hourswith shaking at 37° C. The cells are harvested by centrifugation andinclusion bodies isolated as described in WO 94/00571. The expression ofAPT635 was determined by SDS-PAGE and confirmed by the appearance of anovel protein species that migrated at approximately 8000 Da.

Example 30 A Method for the Preparation of Recombinant Human CD59 with aC-terminal Cysteine, Expressed in Baculovirus/Insect Cells (APT2060)

APT2060 is a protein that comprises soluble human CD59 and a C-terminalcysteine residue following codon 81 of the mature CD59 protein (SEQ IDNO: 40) The protein was expressed in a recombinant form in a baculovirusexpression system. The polymerase chain reaction was used to produce atruncated cDNA encoding soluble CD59 from a full length cDNA (Davies etal. J. Exp. Med. 170, 637, 1989). The 5′ oligonucleotide wascomplementary to 20 bases of the first 7 codons at the N-terminus of themature CD59 protein, and the 3′ oligonucleotide introduced a cysteinecodon and a termination codon immediately following the codon for Ser-81of the mature CD59 protein. These oligonucleotides were also designed tocontain recognition sequences for restriction endonucleases compatiblewith the polylinker site of pBacPAK 8 baculovirus transfer vector(Clontech). The DNA fragment resulting from the PCR amplification wasthen ligated into pBacPAK 8 DNA. This plasmid was then transfected intoSf9 cells with Bacfectin (Clontech) and BacPAK6 viral DNA which had beencut with the restriction endonuclease Bsu36I. This mixture was depositedonto a 50% confluent monolayer of Sf9 cells and left at 28° C. for 3days. The supernatant was removed and a plaque assay performed on serialdilutions of the transfection supernatant as described in BaculovirusExpression Protocols, Methods in Molecular Biology series, ed. C.Richardson). Individual plaques were then picked into 0.5 mL IPL-41medium (Gibco BRL) containing 1% foetal calf serum. The mixture was leftat room temperature for 15 minutes and 100 ml of this solution used toinoculate a 50% confluent monolayer of Sf9 cells. The cells were thenleft to become infected for 4-5 days at 28° C. After this time, thesupernatant was removed and assayed for CD59 expression by Western blotas described in methods. For scale-up of the recombinant virus, thesupernatant was used as an inoculum to infect more Sf9 cell monolayersas described above; alternatively, the supernatant can be used to infectSf9 cells grown in suspension cultures. In this method, 100 mL Sf9 cellsat a concentration of 5×10⁶ cells/ml in IPL-41 medium containing 1% FCSwere inoculated with 50 ml of viral supernatant. The culture was shakenfor 5-7 days at 27° C. and cells removed by centrifugation. Therecombinant virus may be stored at 4° C. until use. APT2060 may bedetected by Western blot as described in Methods and purified using anaffinity column as described.

Example 31 A Method for the Preparation of Recombinant Human CD59 with aC-terminal Cysteine, Expressed in Chinese Hamster Ovary Cells (APT2061;SEQ ID. NO: 42)

APT2061 is a protein that comprises soluble human CD59 and a C-terminalcysteine residue at position 71 of the mature protein. The protein maybe expressed in a recombinant form in chinese hamster ovary cells asdescribed in Methods. Briefly, the polymerase chain reaction is used toproduce a truncated cDNA encoding soluble CD59 from a full length cDNA(Davies et al. J. Exp. Med. 170, 637, 1989). The 5′ oligonucleotide iscomplementary to the first codons at the N-terminus of the mature CD59protein, and the 3′ oligonucleotide introduces a cysteine codon and atermination codon immediately following the codon for Asn-70 of the CD59cDNA. These oligonucleotides can also designed to contain recognitionsequences for restriction endonucleases compatible with the polylinkersite of a CHO expression vector, as described.

Example 32 A Method for the Conjugation of APT542 to APT633, APT635,APT2060 or APT2061 to Generate Compounds APT2062 (Conjugate of SEQ IDNO: 41 and the Base Peptide of SEQ ID NO: 5), APT2063 (Conjugate of SEQID NO: 40 and the Base Peptide of SEQ ID NO: 5), APT2064 (Also aConjugate of SEQ ID NO: 41 and the Base Peptide of SEQ ID NO: 5) andAPT2065 (Conjugate of SEQ ID NO: 42 and the Base Peptide of SEQ ID NO:5)

Compounds APT2062, APT2063, APT2064 and APT2065 are generated bytreating their compounds APT633, APT635, APT2060 and APT2061 with asingle molar equivalent of tris-2-carboxyethyl phosphine (TCEP; in 10 mMHepes, pH7.4) overnight at room temperature. To this mixture is added asolution containing 5 molar equivalents of APT542 (MSWP-1) for 2 hoursat room temperature.

Example 33 A Method for th Synthesis and Characterization of APT2057(SEQ ID NO: 48)

APT2057 is a protein that comprises the short consensus repeats 2,3 and4 of human CD55 (decay accelerating factor, DAF), with a carboxylterminal cysteine residue and an amino terminal histidine tag motifexpressed in a recombinant form in E. coli cells. cDNA to human DAF mRNAwas generated from total brain RNA (OriGene Technologies, USA). Reversetranscription was primed with 40 ρmol of primer DAF-R(5′GGAATTCTAAGTCAGCAAGCCCATGGTTACT 3′) (SEQ ID NO: 49), 3 μg human braintotal RNA and other reagents as recommended by the the RT systemmanufacturers (Promega, Southampton, UK). Half of the RT reaction (10μl) was used as template for PCR. Reaction volume was increased to 50 μlby the addition of water, buffer, MgCl₂ (to 2 mM), DMSO (to 5%) and 20ρmol oligonucleotide DAF-F. (5′GCATATGACCGTCGCGCGGCCGAGC 3′) (SEQ ID NO:50). One unit of Taq polymerase (MBI Fermentas, Vilnius, Lithuania) wasadded, and the reaction subjected to 35 cycles of PCR (94° C., 30 sec;64° C., 30 sec; 72° C., 60 sec). A PCR product of 1156 bp was identifiedby agarose gel electrophoresis, purified from the gel and ligated usingstandard procedures into the T-cloning vector pUC57/T (MBI-Fermentas,Vilnius, Lithuania). Positive clones were identified by PCR screen,analysed by plasmid restriction map and confirmed by full sequenceanalysis. A plasmid to encode APT2057 was generated by PCR using thepUC-DAF plasmid as template. Primers were designed to amplify the regionof the DAF gene encoding amino acids 97-285 (SCR2-4). The 5′ primerincorporated an Ndel restriction enzyme site, and a codon specifyingglutamine, thereby introducing an amino terminal methionine-glutamineamino acid pair. The 3′ primer added a carboxyl terminal cysteineresidue and incorporated an EcoRI restriction enzyme site. The PCRproduct was cloned into the pUC57/T T-vector as described, sequenced,the insert excised with Ndel and EcoRI, and ligated into pET15b(Novagen, Madison, USA, see Methods section). The product of thisligation is the plasmid pET100-02, which expresses DAF(SCR2-4) as anin-frame fusion of a 20 amino acid leader sequence(MGSSHHHHHHSSGLVPRGSH) (SEQ ID NO: 48) to the 191 amino acid DAFSCRs2-4. pET100-02 DNA was introduced into E. coli HAMS113 andtransformed cells selected by virtue of their ability to grow on LB+agarplates in the presence of 50 μg/ml ampicillin (LBAMP). A single colonyrepresenting HAMS113 containing DNA with the coding capacity for APT2057was grown overnight at 37° C. with shaking (200 rpm) in LBAMP medium,then diluted 1:100 into 1 liter fresh LBAMP and growth at 37° C. withshaking. Growth was monitored by measurement of culture turbidity at 600nm, and upon reaching an optical density of 0.6, isopropylβ-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM, followed by a further 3 hours of growth under the same conditions asdescribed above. The expression of APT2057 was analysed by SDS-PAGE(described in methods). APT2057 appeared as a unique protein product ofapproximately 24000 Da as estimated by comparative mobility withmolecular weight standards. Cells containing APT2057 are harvested bycentrifugation and inclusion bodies isolated as follows. Briefly, thecells are resuspended in lysis buffer (50 mM Tris, 1 mM ethylene diaminetetra-acetic acid (ETDA), 50 mM NaCl, pH 8.0) at 50 ml per liter ofinitial culture. The suspension is lysed by two passages through anEmulsiflex homogensier (Glen-Creston, Middlesex UK), followed bycentrifugation at 15000×g to purify inclusion bodies. Inclusion bodiesare initially resuspended to approximately 1 mg.ml⁻¹ (as estimated fromSDS-PAGE) in 20 mM Tris, 1 mM EDTA. 50 mM 2-mercaptoethanol, pH8.5, andsubsequently diluted to a final concentration of 8M urea by the additionof 10 M urea 20 mM Tris, 1 mM EDTA, 50 mM 2-mercaptoethanol, pH8.5. Thissuspension is stirred at 4° C. for 16 hours, and insoluble materialremoved by centrifugation at 15000×g for 30 minutes. The APT2057 isrefolded by 1 in 50 dilution into 20 mM ethanolamine, 1 mM EDTA, pH 11buffer and static incubation at 4° C. for 24 hours. Insoluble materialis removed by centrifugation (10000×g, 10 minutes), and soluble materialbuffer exchanged into Dulbecco's A PBS, pH 7.4 using an XK50×23 cmSephadex G25 column. Refolded APT205B is analyzed by SDS-PAGE, Westernblot and the effectiveness of the protein in a haemolytic assay(described in methods).

Example 34 A Method for the Synthesis and Characterization of APT2058(SEQ ID NO: 47)

APT2058 is a protein that comprises the short consensus repeats 1,2,3and 4 of human CD55 (decay accelerating factor, DAF), with a carboxylterminal cysteine residue and an amino terminal histidine tag motifexpressed in a recombinant form in E. coli cells. cDNA to human DAF mRNAwas generated from total brain RNA as described in Example 9. A plasmidto encode APT2058 was generated by PCR using the pUC-DAF plasmid astemplate. Primers were designed to amplify the region of the DAF geneencoding amino acids 35-285 (SCR1-4). The 5′ primer incorporated an Ndelrestriction enzyme site, and a codon specifying glutamine, therebyintroducing an amino terminal methionine-glutamine amino acid pair. The3′ primer added a carboxyl terminal cysteine residue and incorporated anEcoRI restriction enzyme site. The PCR product was cloned into thepUC57/T T-vector as described, sequenced, the insert excised with Ndeland EcoRI, and ligated into pET15b (Novagen, Madison, USA). The productof this ligation is the plasmid pET99-02, which expresses DAF (SCR1-4)as an in-frame fusion of a 20 amino acid leader sequence(MGSSHHHHHHSSGLVPRGSH) (SEQ ID NO: 48) to the 251 amino acid DAF SCRs1-4(APT2058). pET99-02 DNA was introduced into E. coli HAMS 113 (seemethods) and expression of the recombinant protein induced as describedin Example 1. The expression of APT2058 was analysed by SDS-PAGE(described in methods). APT2058 appeared as a unique protein product ofapproximately 31000 Da as estimated by comparative mobility withmolecular weight standards. Cells containing APT2058 were harvested bycentrifugation and inclusion bodies isolated as follows. Briefly, thecells were resuspended in lysis buffer (50 mM Tris, 1 mM ethylenediamine tetra-acetic acid (ETDA), 50 mM NaCl, pH 8.0) at 50 ml per literof initial culture. The suspension was lysed by two passages through anEmulsiflex homogensier (Glen-Creston, Middlesex, UK), followed bycentrifugation at 15000×g to purify inclusion bodies. Inclusion bodieswere initially resuspended to approximately 1 mg.ml⁻¹ (as estimated fromSDS-PAGE) in 20 mM Tris, 1 mM EDTA, 50 mM 2-mercaptoethanol, pH8.5, andsubsequently diluted to a final concentration of 8M urea by the additionof 10 M urea 20 mM Tris, 1 mM EDTA, 50 mM 2-mercaptoethanol, pH8.5. Thissuspension was stirred at 4° C. for 16 hours, and insoluble materialremoved by centrifugation at 15000×g for 30 minutes. The APT2057 wasrefolded by 1 in 50 dilution into 20 mM ethanolamine, 1 mM EDTA, pH 11buffer and static incubation at 4° C. for 24 hours. Insoluble materialwas removed by centrifugation (10000×g, 10 minutes), and solublematerial buffer exchanged into Dulbecco's A PBS, pH 7.4 using an XK50×23cm Sephadex G25 column. Refolded APT2058 was analysed by SDS-PAGE,Western blot and the effectiveness of the protein in a haemolytic assay(described in methods). Using this assay (at 1:400 dilution of humanserum), the concentration of APT2058 required to bring about 50%inhibition of lysis (IH₅₀) was approximately 3 nM.

Example 35 A Method for the Synthesis and Characterization of APT2160(Conjugate of SEQ ID NO: 47 and the Base Peptide of SEQ ID NO: 5)

Compound APT2160 was generated by treating the parent compound APT2058(at approximately 100 μM) with a three-fold molar excess of 10 mMtris-2-carboxyethyl phosphine (TCEP: in 50 mM Hepes, pH 4.5) overnightat room temperature. To this mixture was added a solution containingfive molar equivalents of MSWP-1 (Example 2) in 100% DMSO for 2 hours atroom temperature. APT2160 was characterized by observation of a mobilityshift on non-reducing SDS-PAGE of approximately 2000 Da, consistent withthe addition of a single molecule of APT542 to APT2058. The compound wasassayed in the haemolytic assay (at 1:400 dilution of human serum) andan IH₅₀ value of 0.03 nM was found.

Example 36 A Method for the Synthesis and Characterization of APT2184(Conjugate of SEQ ID NO: 46 and the Base Peptide of SEQ ID NO: 5)

Compound APT2184 was generated by treating the parent compound APT2057with a three-fold molar excess of 10 mM tris-2-carboxyethyl phosphine(TCEP: in 50 mM Hepes, pH 4.5) overnight at room temperature. To thismixture is added a solution containing five molar equivalents of MSWP-1in 100% DMSO for 2 hours at room temperature.

It is to be understood that the description, specific examples and data,while indicating exemplary embodiments, are given by way of illustrationand are not intended to limit the present invention. Various changes andmodifications within the present invention will become apparent to theskilled artisan from the discussion, disclosure and data combinedherein, and thus are considered part of the invention.

This application claims priority to GB 9614871.3, filed Jul. 15, 1996,there entirety of which is hereby incorporated by reference.

53 1 37 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 1 gcaccgcagt gcatcatccc gaacaaatgc taataaa 372 37 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 2 agcttttatt agcatttgtt cgggatgatg cactgcg 373 85 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 3 gcaccgcagt gcatcatccc gaacaaagac ggtccgaaaaagaagaaaaa gaaatctccg 60 tccaaatctt ccggttgcta ataaa 85 4 85 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 4 agcttttatt agcaaccgga agatttggac ggagatttct ttttcttctttttcggaccg 60 tctttgttcg ggatgatgca ctgcg 85 5 17 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide used tosynthesize MSWP-1 5 Gly Ser Ser Lys Ser Pro Ser Lys Lys Lys Lys Lys LysPro Gly Asp 1 5 10 15 Cys 6 198 PRT Artificial Sequence Description ofArtificial Sequence [SCR1-3]- Cys protein 6 Met Gln Cys Asn Ala Pro GluTrp Leu Pro Phe Ala Arg Pro Thr Asn 1 5 10 15 Leu Thr Asp Glu Phe GluPhe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu 20 25 30 Cys Arg Pro Gly Tyr SerGly Arg Pro Phe Ser Ile Ile Cys Leu Lys 35 40 45 Asn Ser Val Trp Thr GlyAla Lys Asp Arg Cys Arg Arg Lys Ser Cys 50 55 60 Arg Asn Pro Pro Asp ProVal Asn Gly Met Val His Val Ile Lys Gly 65 70 75 80 Ile Gln Phe Gly SerGln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg 85 90 95 Leu Ile Gly Ser SerSer Ala Thr Cys Ile Ile Ser Gly Asp Thr Val 100 105 110 Ile Trp Asp AsnGlu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu 115 120 125 Pro Pro ThrIle Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn 130 135 140 Phe HisTyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly 145 150 155 160Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr 165 170175 Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys 180185 190 Ile Ile Pro Asn Lys Cys 195 7 214 PRT Artificial SequenceDescription of Artificial Sequence [SCR1-3]/ switch fusion protein 7 MetGln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn 1 5 10 15Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu 20 25 30Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys 35 40 45Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys 50 55 60Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly 65 70 7580 Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg 85 9095 Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val 100105 110 Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu115 120 125 Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg GluAsn 130 135 140 Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro GlySer Gly 145 150 155 160 Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro SerIle Tyr Cys Thr 165 170 175 Ser Asn Asp Asp Gln Val Gly Ile Trp Ser GlyPro Ala Pro Gln Cys 180 185 190 Ile Ile Pro Asn Lys Asp Gly Pro Lys LysLys Lys Lys Lys Ser Pro 195 200 205 Ser Lys Ser Ser Gly Cys 210 8 16 PRTArtificial Sequence Description of Artificial Sequence Illustrativeamino acid sequence 8 Asp Gly Pro Lys Lys Lys Lys Lys Lys Ser Pro SerLys Ser Ser Gly 1 5 10 15 9 16 PRT Artificial Sequence Description ofArtificial Sequence Illustrative amino acid sequence 9 Gly Ser Ser LysSer Pro Ser Lys Lys Lys Lys Lys Lys Pro Gly Asp 1 5 10 15 10 20 PRTArtificial Sequence Description of Artificial Sequence Illustrativeamino acid sequence 10 Ser Pro Ser Asn Glu Thr Pro Lys Lys Lys Lys LysArg Phe Ser Phe 1 5 10 15 Lys Lys Ser Gly 20 11 16 PRT ArtificialSequence Description of Artificial Sequence Illustrative amino acidsequence 11 Asp Gly Pro Lys Lys Lys Lys Lys Lys Ser Pro Ser Lys Ser SerLys 1 5 10 15 12 14 PRT Artificial Sequence Description of ArtificialSequence Illustrative amino acid sequence 12 Ser Lys Asp Gly Lys Lys LysLys Lys Lys Ser Lys Thr Lys 1 5 10 13 6 PRT Artificial SequenceDescription of Artificial Sequence Illustrative amino acid sequence 13Gly Arg Gly Asp Ser Pro 1 5 14 209 PRT Artificial Sequence Descriptionof Artificial Sequence SCR1-3 with the c-terminal amino acids N195 andK196 replaced by a 14 amino acid peptide 14 Met Gln Cys Asn Ala Pro GluTrp Leu Pro Phe Ala Arg Pro Thr Asn 1 5 10 15 Leu Thr Asp Glu Phe GluPhe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu 20 25 30 Cys Arg Pro Gly Tyr SerGly Arg Pro Phe Ser Ile Ile Cys Leu Lys 35 40 45 Asn Ser Val Trp Thr GlyAla Lys Asp Arg Cys Arg Arg Lys Ser Cys 50 55 60 Arg Asn Pro Pro Asp ProVal Asn Gly Met Val His Val Ile Lys Gly 65 70 75 80 Ile Gln Phe Gly SerGln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg 85 90 95 Leu Ile Gly Ser SerSer Ala Thr Cys Ile Ile Ser Gly Asp Thr Val 100 105 110 Ile Trp Asp AsnGlu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu 115 120 125 Pro Pro ThrIle Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn 130 135 140 Phe HisTyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly 145 150 155 160Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr 165 170175 Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys 180185 190 Ile Ile Pro Thr Asn Ala Asn Lys Ser Leu Ser Ser Ile Ser Cys Gln195 200 205 Thr 15 53 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 15 ctggagcggg cccgcaccgc agtgcatcatcccgaacaaa tgctaataaa agc 53 16 53 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic oligonucleotide 16 gcttttattagcatttgttc gggatgatgc actgcggtgc gggcccgctc cag 53 17 13 PRT ArtificialSequence Description of Artificial Sequence Illustrative amino acidsequence 17 Asp Gly Pro Ser Glu Ile Leu Arg Gly Asp Phe Ser Ser 1 5 1018 17 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide used to generate MSWP-2 18 Cys Asp Gly Pro Lys Lys LysLys Lys Lys Ser Pro Ser Lys Ser Ser 1 5 10 15 Lys 19 15 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide used togenerate MSWP-3 19 Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys Ser Lys ThrLys Cys 1 5 10 15 20 16 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide used to generate TCTP-1 20 Cys SerAla Ala Pro Ser Ser Gly Phe Arg Ile Leu Leu Leu Lys Val 1 5 10 15 21 17PRT Artificial Sequence Description of Artificial Sequence Illustrativeamino acid sequence 21 Gly Asn Glu Gln Ser Phe Arg Val Asp Leu Arg ThrLeu Leu Arg Tyr 1 5 10 15 Ala 22 9 PRT Artificial Sequence Descriptionof Artificial Sequence Illustrative amino acid sequence 22 Gly Phe ArgIle Leu Leu Leu Lys Val 1 5 23 211 PRT Artificial Sequence Descriptionof Artificial Sequence SCR1-3 with an additional 14 amino acid residuesat the c-terminus 23 Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala ArgPro Thr Asn 1 5 10 15 Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr TyrLeu Asn Tyr Glu 20 25 30 Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser IleIle Cys Leu Lys 35 40 45 Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys ArgArg Lys Ser Cys 50 55 60 Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val HisVal Ile Lys Gly 65 70 75 80 Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser CysThr Lys Gly Tyr Arg 85 90 95 Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile IleSer Gly Asp Thr Val 100 105 110 Ile Trp Asp Asn Glu Thr Pro Ile Cys AspArg Ile Pro Cys Gly Leu 115 120 125 Pro Pro Thr Ile Thr Asn Gly Asp PheIle Ser Thr Asn Arg Glu Asn 130 135 140 Phe His Tyr Gly Ser Val Val ThrTyr Arg Cys Asn Pro Gly Ser Gly 145 150 155 160 Gly Arg Lys Val Phe GluLeu Val Gly Glu Pro Ser Ile Tyr Cys Thr 165 170 175 Ser Asn Asp Asp GlnVal Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys 180 185 190 Ile Ile Pro AsnLys Asp Gly Pro Ser Glu Ile Leu Arg Gly Asp Phe 195 200 205 Ser Ser Cys210 24 15 PRT Artificial Sequence Description of Artificial SequenceIllustrative amino acid sequence 24 Ser Ala Ala Pro Ser Ser Gly Phe ArgIle Leu Leu Leu Lys Val 1 5 10 15 25 72 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 25cgcaccgcag tgcatcatcc cgaacaaaga tggcccgagc gaaattctgc gtggcgattt 60tagcagctgc ta 72 26 80 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 26 acgttagcag ctgctaaaat cgccacgcagaatttcgctc gggccatctt tgttcgggat 60 gatgcactgc ggtgcgggcc 80 27 17 PRTArtificial Sequence Description of Artificial Sequence Myristoyl/Electrostatic Swith Peptide Reagent 1 (MSWP-1) 27 Gly Ser Ser Lys SerPro Ser Lys Lys Lys Lys Lys Lys Pro Gly Asp 1 5 10 15 Cys 28 17 PRTArtificial Sequence Description of Artificial Sequence Myristoyl/Electrostatic Switch Peptide Reagent 2 (MSWP-2) 28 Cys Asp Gly Pro LysLys Lys Lys Lys Lys Ser Pro Ser Lys Ser Ser 1 5 10 15 Lys 29 15 PRTArtificial Sequence Description of Artificial Sequence Myristoyl/Electrostatic Switch Peptide Reagent 3 (MSWP-3) 29 Ser Lys Asp Gly LysLys Lys Lys Lys Lys Ser Lys Thr Lys Cys 1 5 10 15 30 16 PRT ArtificialSequence Description of Artificial Sequence T-cell targeting peptidereagent 1 (TCTP-1) 30 Cys Ser Ala Ala Pro Ser Ser Gly Phe Arg Ile LeuLeu Leu Lys Val 1 5 10 15 31 214 PRT Artificial Sequence Description ofArtificial Sequence [SCR1-3/ switch fusion]-[MAET] 31 Met Gln Cys AsnAla Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn 1 5 10 15 Leu Thr AspGlu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu 20 25 30 Cys Arg ProGly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys 35 40 45 Asn Ser ValTrp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys 50 55 60 Arg Asn ProPro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly 65 70 75 80 Ile GlnPhe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg 85 90 95 Leu IleGly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val 100 105 110 IleTrp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu 115 120 125Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn 130 135140 Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly 145150 155 160 Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr CysThr 165 170 175 Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala ProGln Cys 180 185 190 Ile Ile Pro Asn Lys Asp Gly Pro Lys Lys Lys Lys LysLys Ser Pro 195 200 205 Ser Lys Ser Ser Gly Cys 210 32 17 PRT ArtificialSequence Description of Artificial Sequence Illustrative amino acidsequence 32 Ala Ala Pro Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys ValAla 1 5 10 15 Gly 33 215 PRT Artificial Sequence Description ofArtificial Sequence SCR1-3 with an additional c-terminal 18 amino acids33 Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn 1 510 15 Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu 2025 30 Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys 3540 45 Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys 5055 60 Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly 6570 75 80 Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg85 90 95 Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val100 105 110 Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys GlyLeu 115 120 125 Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn ArgGlu Asn 130 135 140 Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn ProGly Ser Gly 145 150 155 160 Gly Arg Lys Val Phe Glu Leu Val Gly Glu ProSer Ile Tyr Cys Thr 165 170 175 Ser Asn Asp Asp Gln Val Gly Ile Trp SerGly Pro Ala Pro Gln Cys 180 185 190 Ile Ile Pro Asn Lys Ala Ala Pro SerVal Ile Gly Phe Arg Ile Leu 195 200 205 Leu Leu Lys Val Ala Gly Cys 210215 34 84 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 34 cgcaccgcag tgcatcatcc cgaacaaagc ggcgcccagcgtgattggct tccgtattct 60 gctgctgaaa gtggcgggct gcta 84 35 92 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 35 agcttagcag cccgccactt tcagcagcag aatacggaagccaatcacgc tgggcgccgc 60 tttgttcggg atgatgcact gcggtgcggg cc 92 36 17PRT Artificial Sequence Description of Artificial Sequence Illustrativeamino acid sequence 36 Asp Gly Pro Lys Lys Lys Lys Lys Lys Ser Pro SerLys Ser Ser Gly 1 5 10 15 Cys 37 77 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic protein APT631 37 Leu Gln Cys Tyr AsnCys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys SerSer Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val TyrAsn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn 35 40 45 Asp Val Thr ThrArg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp LeuCys Asn Phe Asn Glu Gln Leu Glu Asn 65 70 75 38 17 PRT ArtificialSequence Description of Artificial Sequence Synthetic protein APT542 38Gly Ser Ser Lys Ser Pro Ser Lys Lys Lys Lys Lys Lys Pro Gly Asp 1 5 1015 Cys 39 70 PRT Artificial Sequence Description of Artificial SequenceSynthetic protein APT634 39 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr AlaAsp Cys Lys Thr Ala 1 5 10 15 Val Ala Cys Ser Ser Asp Phe Asp Ala CysLeu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys PheGlu His Cys Asn Phe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn GluLeu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn 65 70 40 82 PRTArtificial Sequence Description of Artificial Sequence Synthetic proteinAPT2060 40 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys ThrAla 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr LysAla Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys AsnPhe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr TyrCys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn GlyGly Thr 65 70 75 80 Ser Cys 41 83 PRT Artificial Sequence Description ofArtificial Sequence Synthetic protein APT635 41 Met Leu Gln Cys Tyr AsnCys Pro Asn Pro Thr Ala Asp Cys Lys Thr 1 5 10 15 Ala Val Asn Cys SerSer Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala 20 25 30 Gly Leu Gln Val TyrAsn Lys Cys Trp Lys Phe Glu His Cys Asn Phe 35 40 45 Asn Asp Val Thr ThrArg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys 50 55 60 Cys Lys Lys Asp LeuCys Asn Phe Asn Glu Gln Leu Glu Asn Gly Gly 65 70 75 80 Thr Ser Cys 4271 PRT Artificial Sequence Description of Artificial Sequence Syntheticprotein APT2061 42 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp CysLys Thr Ala 1 5 10 15 Val Ala Cys Ser Ser Asp Phe Asp Ala Cys Leu IleThr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu HisCys Asn Phe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu ThrTyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Cys 65 70 43 18 PRTArtificial Sequence Description of Artificial Sequence Illustrativeamino acid sequence 43 Ala Ala Pro Ser Val Ile Gly Phe Arg Ile Leu LeuLeu Lys Val Ala 1 5 10 15 Gly Cys 44 14 PRT Artificial SequenceDescription of Artificial Sequence Illustrative amino acid sequence 44Asp Gly Pro Ser Glu Ile Leu Arg Gly Asp Phe Ser Ser Cys 1 5 10 45 36 DNAArtificial Sequence Description of Artificial Sequence Illustrativeoligonucleotide 45 cctctggcca aatgtacctc tcgtgcacat tgctga 36 46 211 PRTArtificial Sequence Description of Artificial Sequence Synthetic proteinAPT2057 46 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu ValPro 1 5 10 15 Arg Gly Ser His Met Ser Cys Glu Val Pro Thr Arg Leu AsnSer Ala 20 25 30 Ser Leu Lys Gln Pro Tyr Ile Thr Gln Asn Tyr Phe Pro ValGly Thr 35 40 45 Val Val Glu Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu ProSer Leu 50 55 60 Ser Pro Lys Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser ThrAla Val 65 70 75 80 Glu Phe Cys Lys Lys Lys Ser Cys Pro Asn Pro Gly GluIle Arg Asn 85 90 95 Gly Gln Ile Asp Val Pro Gly Gly Ile Leu Phe Gly AlaThr Ile Ser 100 105 110 Phe Ser Cys Asn Thr Gly Tyr Lys Leu Phe Gly SerThr Ser Ser Phe 115 120 125 Cys Leu Ile Ser Gly Ser Ser Val Gln Trp SerAsp Pro Leu Pro Glu 130 135 140 Cys Arg Glu Ile Tyr Cys Pro Ala Pro ProGln Ile Asp Asn Gly Ile 145 150 155 160 Ile Gln Gly Glu Arg Asp His TyrGly Tyr Arg Gln Ser Val Thr Tyr 165 170 175 Ala Cys Asn Lys Gly Phe ThrMet Ile Gly Glu His Ser Ile Tyr Cys 180 185 190 Thr Val Asn Asn Asp GluGly Glu Trp Ser Gly Pro Pro Pro Glu Cys 195 200 205 Arg Gly Cys 210 47274 PRT Artificial Sequence Description of Artificial Sequence Syntheticprotein APT2058 47 Met Gly Ser Ser His His His His His His Ser Ser GlyLeu Val Pro 1 5 10 15 Arg Gly Ser His Met Gln Asp Cys Gly Leu Pro ProAsp Val Pro Asn 20 25 30 Ala Gln Pro Ala Leu Glu Gly Arg Thr Ser Phe ProGlu Asp Thr Val 35 40 45 Ile Thr Tyr Lys Cys Glu Glu Ser Phe Val Lys IlePro Gly Glu Lys 50 55 60 Asp Ser Val Ile Cys Leu Lys Gly Ser Gln Trp SerAsp Ile Glu Glu 65 70 75 80 Phe Cys Asn Arg Ser Cys Glu Val Pro Thr ArgLeu Asn Ser Ala Ser 85 90 95 Leu Lys Gln Pro Tyr Ile Thr Gln Asn Tyr PhePro Val Gly Thr Val 100 105 110 Val Glu Tyr Glu Cys Arg Pro Gly Tyr ArgArg Glu Pro Ser Leu Ser 115 120 125 Pro Lys Leu Thr Cys Leu Gln Asn LeuLys Trp Ser Thr Ala Val Glu 130 135 140 Phe Cys Lys Lys Lys Ser Cys ProAsn Pro Gly Glu Ile Arg Asn Gly 145 150 155 160 Gln Ile Asp Val Pro GlyGly Ile Leu Phe Gly Ala Thr Ile Ser Phe 165 170 175 Ser Cys Asn Thr GlyTyr Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys 180 185 190 Leu Ile Ser GlySer Ser Val Gln Trp Ser Asp Pro Leu Pro Glu Cys 195 200 205 Arg Glu IleTyr Cys Pro Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile 210 215 220 Gln GlyGlu Arg Asp His Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala 225 230 235 240Cys Asn Lys Gly Phe Thr Met Ile Gly Glu His Ser Ile Tyr Cys Thr 245 250255 Val Asn Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg 260265 270 Gly Cys 48 20 PRT Artificial Sequence Description of ArtificialSequence Synthetic leader sequence 48 Met Gly Ser Ser His His His HisHis His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His 20 49 31 DNAArtificial Sequence Description of Artificial Sequence Primer DAF- R 49ggaattctaa gtcagcaagc ccatggttac t 31 50 25 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide DAF-F 50gcatatgacc gtcgcgcggc cgagc 25 51 527 PRT Homo sapiens tissueplasminogen activator 51 Ser Tyr Gln Val Ile Cys Arg Asp Glu Lys Thr GlnMet Ile Tyr Gln 1 5 10 15 Gln His Gln Ser Trp Leu Arg Pro Val Leu ArgSer Asn Arg Val Glu 20 25 30 Tyr Cys Trp Cys Asn Ser Gly Arg Ala Gln CysHis Ser Val Pro Val 35 40 45 Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn GlyGly Thr Cys Gln Gln 50 55 60 Ala Leu Tyr Phe Ser Asp Phe Val Cys Gln CysPro Glu Gly Phe Ala 65 70 75 80 Gly Lys Cys Cys Glu Ile Asp Thr Arg AlaThr Cys Tyr Glu Asp Gln 85 90 95 Gly Ile Ser Tyr Arg Gly Thr Trp Ser ThrAla Glu Ser Gly Ala Glu 100 105 110 Cys Thr Asn Trp Asn Ser Ser Ala LeuAla Gln Lys Pro Tyr Ser Gly 115 120 125 Arg Arg Pro Asp Ala Ile Arg LeuGly Leu Gly Asn His Asn Tyr Cys 130 135 140 Arg Asn Pro Asp Arg Asp SerLys Pro Trp Cys Tyr Val Phe Lys Ala 145 150 155 160 Gly Lys Tyr Ser SerGlu Phe Cys Ser Thr Pro Ala Cys Ser Glu Gly 165 170 175 Asn Ser Asp CysTyr Phe Gly Asn Gly Ser Ala Tyr Arg Gly Thr His 180 185 190 Ser Leu ThrGlu Ser Gly Ala Ser Cys Leu Pro Trp Asn Ser Met Ile 195 200 205 Leu IleGly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala Gln Ala Leu 210 215 220 GlyLeu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys 225 230 235240 Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys 245250 255 Asp Val Pro Ser Cys Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro260 265 270 Gln Phe Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser HisPro 275 280 285 Trp Gln Ala Ala Ile Phe Ala Lys His Arg Arg Ser Pro GlyGlu Arg 290 295 300 Phe Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp IleLeu Ser Ala 305 310 315 320 Ala His Cys Phe Gln Glu Arg Phe Pro Pro HisHis Leu Thr Val Ile 325 330 335 Leu Gly Arg Thr Tyr Arg Val Val Pro GlyGlu Glu Glu Gln Lys Phe 340 345 350 Glu Val Glu Lys Tyr Ile Val His LysGlu Phe Asp Asp Asp Thr Tyr 355 360 365 Asp Asn Asp Ile Ala Leu Leu GlnLeu Lys Ser Asp Ser Ser Arg Cys 370 375 380 Ala Gln Glu Ser Ser Val ValArg Thr Val Cys Leu Pro Pro Ala Asp 385 390 395 400 Leu Gln Leu Pro AspTrp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys 405 410 415 His Glu Ala LeuSer Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His 420 425 430 Val Arg LeuTyr Pro Ser Ser Arg Cys Thr Ser Gln His Leu Leu Asn 435 440 445 Arg ThrVal Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly 450 455 460 GlyPro Gln Ala Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly 465 470 475480 Pro Leu Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile Ile 485490 495 Ser Trp Gly Leu Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr Thr500 505 510 Lys Val Thr Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met Arg Pro515 520 525 52 1947 PRT Homo sapiens CR1 52 Gln Cys Asn Ala Pro Glu TrpLeu Pro Phe Ala Arg Pro Thr Asn Leu 1 5 10 15 Thr Asp Glu Phe Glu PhePro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys 20 25 30 Arg Pro Gly Tyr Ser GlyArg Pro Phe Ser Ile Ile Cys Leu Lys Asn 35 40 45 Ser Val Trp Thr Gly AlaLys Asp Arg Cys Arg Arg Lys Ser Cys Arg 50 55 60 Asn Pro Pro Asp Pro ValAsn Gly Met Val His Val Ile Lys Gly Ile 65 70 75 80 Gln Phe Gly Ser GlnIle Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu 85 90 95 Ile Gly Ser Ser SerAla Thr Cys Ile Ile Ser Gly Asp Thr Val Ile 100 105 110 Trp Asp Asn GluThr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro 115 120 125 Pro Thr IleThr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe 130 135 140 His TyrGly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly 145 150 155 160Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser 165 170175 Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile 180185 190 Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile Leu Val195 200 205 Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu PheArg 210 215 220 Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val LysCys Gln 225 230 235 240 Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser CysSer Arg Val Cys 245 250 255 Gln Pro Pro Pro Asp Val Leu His Ala Glu ArgThr Gln Arg Asp Lys 260 265 270 Asp Asn Phe Ser Pro Gly Gln Glu Val PheTyr Ser Cys Glu Pro Gly 275 280 285 Tyr Asp Leu Arg Gly Ala Ala Ser MetArg Cys Thr Pro Gln Gly Asp 290 295 300 Trp Ser Pro Ala Ala Pro Thr CysGlu Val Lys Ser Cys Asp Asp Phe 305 310 315 320 Met Gly Gln Leu Leu AsnGly Arg Val Leu Phe Pro Val Asn Leu Gln 325 330 335 Leu Gly Ala Lys ValAsp Phe Val Cys Asp Glu Gly Phe Gln Leu Lys 340 345 350 Gly Ser Ser AlaSer Tyr Cys Val Leu Ala Gly Met Glu Ser Leu Trp 355 360 365 Asn Ser SerVal Pro Val Cys Glu Gln Ile Phe Cys Pro Ser Pro Pro 370 375 380 Val IlePro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val Phe Pro 385 390 395 400Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp Arg Gly 405 410415 Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr Ser Asp 420425 430 Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys Gly Ile435 440 445 Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys LeuLys 450 455 460 Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser LeuLys Tyr 465 470 475 480 Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe SerIle Thr Cys Leu 485 490 495 Asp Asn Leu Val Trp Ser Ser Pro Lys Asp ValCys Lys Arg Lys Ser 500 505 510 Cys Lys Thr Pro Pro Asp Pro Val Asn GlyMet Val His Val Ile Thr 515 520 525 Asp Ile Gln Val Gly Ser Arg Ile AsnTyr Ser Cys Thr Thr Gly His 530 535 540 Arg Leu Ile Gly His Ser Ser AlaGlu Cys Ile Leu Ser Gly Asn Ala 545 550 555 560 Ala His Trp Ser Thr LysPro Pro Ile Cys Gln Arg Ile Pro Cys Gly 565 570 575 Leu Pro Pro Thr IleAla Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu 580 585 590 Asn Phe His TyrGly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser 595 600 605 Gly Gly ArgLys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys 610 615 620 Thr SerAsn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln 625 630 635 640Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val Glu Asn Gly Ile 645 650655 Leu Val Ser Asp Asn Arg Ser Leu Phe Ser Leu Asn Glu Val Val Glu 660665 670 Phe Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg Val Lys675 680 685 Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys SerArg 690 695 700 Val Cys Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg ThrGln Arg 705 710 715 720 Asp Lys Asp Asn Phe Ser Pro Gly Gln Glu Val PheTyr Ser Cys Glu 725 730 735 Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser MetArg Cys Thr Pro Gln 740 745 750 Gly Asp Trp Ser Pro Ala Ala Pro Thr CysGlu Val Lys Ser Cys Asp 755 760 765 Asp Phe Met Gly Gln Leu Leu Asn GlyArg Val Leu Phe Pro Val Asn 770 775 780 Leu Gln Leu Gly Ala Lys Val AspPhe Val Cys Asp Glu Gly Phe Gln 785 790 795 800 Leu Lys Gly Ser Ser AlaSer Tyr Cys Val Leu Ala Gly Met Glu Ser 805 810 815 Leu Trp Asn Ser SerVal Pro Val Cys Glu Gln Ile Phe Cys Pro Ser 820 825 830 Pro Pro Val IlePro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val 835 840 845 Phe Pro PheGly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp 850 855 860 Arg GlyThr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys Thr 865 870 875 880Ser Asp Pro Gln Gly Asn Gly Val Trp Ser Ser Pro Ala Pro Arg Cys 885 890895 Gly Ile Leu Gly His Cys Gln Ala Pro Asp His Phe Leu Phe Ala Lys 900905 910 Leu Lys Thr Gln Thr Asn Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu915 920 925 Lys Tyr Glu Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser IleThr 930 935 940 Cys Leu Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val CysLys Arg 945 950 955 960 Lys Ser Cys Lys Thr Pro Pro Asp Pro Val Asn GlyMet Val His Val 965 970 975 Ile Thr Asp Ile Gln Val Gly Ser Arg Ile AsnTyr Ser Cys Thr Thr 980 985 990 Gly His Arg Leu Ile Gly His Ser Ser AlaGlu Cys Ile Leu Ser Gly 995 1000 1005 Asn Thr Ala His Trp Ser Thr LysPro Pro Ile Cys Gln Arg Ile Pro 1010 1015 1020 Cys Gly Leu Pro Pro ThrIle Ala Asn Gly Asp Phe Ile Ser Thr Asn 1025 1030 1035 1040 Arg Glu AsnPhe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Leu 1045 1050 1055 GlySer Arg Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile 1060 10651070 Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala1075 1080 1085 Pro Gln Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn ValGlu Asn 1090 1095 1100 Gly Ile Leu Val Ser Asp Asn Arg Ser Leu Phe SerLeu Asn Glu Val 1105 1110 1115 1120 Val Glu Phe Arg Cys Gln Pro Gly PheVal Met Lys Gly Pro Arg Arg 1125 1130 1135 Val Lys Cys Gln Ala Leu AsnLys Trp Glu Pro Glu Leu Pro Ser Cys 1140 1145 1150 Ser Arg Val Cys GlnPro Pro Pro Glu Ile Leu His Gly Glu His Thr 1155 1160 1165 Pro Ser HisGln Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser 1170 1175 1180 CysGlu Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Leu His Cys Thr 1185 11901195 1200 Pro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg Cys Ala Val LysSer 1205 1210 1215 Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly Arg ValLeu Phe Pro 1220 1225 1230 Leu Asn Leu Gln Leu Gly Ala Lys Val Ser PheVal Cys Asp Glu Gly 1235 1240 1245 Phe Arg Leu Lys Gly Ser Ser Val SerHis Cys Val Leu Val Gly Met 1250 1255 1260 Arg Ser Leu Trp Asn Asn SerVal Pro Val Cys Glu His Ile Phe Cys 1265 1270 1275 1280 Pro Asn Pro ProAla Ile Leu Asn Gly Arg His Thr Gly Thr Pro Ser 1285 1290 1295 Gly AspIle Pro Tyr Gly Lys Glu Ile Ser Tyr Thr Cys Asp Pro His 1300 1305 1310Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly Glu Ser Thr Ile Arg 13151320 1325 Cys Thr Ser Asp Pro His Gly Asn Gly Val Trp Ser Ser Pro AlaPro 1330 1335 1340 Arg Cys Glu Leu Ser Val Arg Ala Gly His Cys Lys ThrPro Glu Gln 1345 1350 1355 1360 Phe Pro Phe Ala Ser Pro Thr Ile Pro IleAsn Asp Phe Glu Phe Pro 1365 1370 1375 Val Gly Thr Ser Leu Asn Tyr GluCys Arg Pro Gly Tyr Phe Gly Lys 1380 1385 1390 Met Phe Ser Ile Ser CysLeu Glu Asn Leu Val Trp Ser Ser Val Glu 1395 1400 1405 Asp Asn Cys ArgArg Lys Ser Cys Gly Pro Pro Pro Glu Pro Phe Asn 1410 1415 1420 Gly MetVal His Ile Asn Thr Asp Thr Gln Phe Gly Ser Thr Val Asn 1425 1430 14351440 Tyr Ser Cys Asn Glu Gly Phe Arg Leu Ile Gly Ser Pro Ser Thr Thr1445 1450 1455 Cys Leu Val Ser Gly Asn Asn Val Thr Trp Asp Lys Lys AlaPro Ile 1460 1465 1470 Cys Glu Ile Ile Ser Cys Glu Pro Pro Pro Thr IleSer Asn Gly Asp 1475 1480 1485 Phe Tyr Ser Asn Asn Arg Thr Ser Phe HisAsn Gly Thr Val Val Thr 1490 1495 1500 Tyr Gln Cys His Thr Gly Pro AspGly Glu Gln Leu Phe Glu Leu Val 1505 1510 1515 1520 Gly Glu Arg Ser IleTyr Cys Thr Ser Lys Asp Asp Gln Val Gly Val 1525 1530 1535 Trp Ser SerPro Pro Pro Arg Cys Ile Ser Thr Asn Lys Cys Thr Ala 1540 1545 1550 ProGlu Val Glu Asn Ala Ile Arg Val Pro Gly Asn Arg Ser Phe Phe 1555 15601565 Ser Leu Thr Glu Ile Ile Arg Phe Arg Cys Gln Pro Gly Phe Val Met1570 1575 1580 Val Gly Ser His Thr Val Gln Cys Gln Thr Asn Gly Arg TrpGly Pro 1585 1590 1595 1600 Lys Leu Pro His Cys Ser Arg Val Cys Gln ProPro Pro Glu Ile Leu 1605 1610 1615 His Gly Glu His Thr Leu Ser His GlnAsp Asn Phe Ser Pro Gly Gln 1620 1625 1630 Glu Val Phe Tyr Ser Cys GluPro Ser Tyr Asp Leu Arg Gly Ala Ala 1635 1640 1645 Ser Leu His Cys ThrPro Gln Gly Asp Trp Ser Pro Glu Ala Pro Arg 1650 1655 1660 Cys Thr ValLys Ser Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly 1665 1670 1675 1680Arg Val Leu Leu Pro Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe 16851690 1695 Val Cys Asp Glu Gly Phe Arg Leu Lys Gly Arg Ser Ala Ser HisCys 1700 1705 1710 Val Leu Ala Gly Met Lys Ala Leu Trp Asn Ser Ser ValPro Val Cys 1715 1720 1725 Glu Gln Ile Phe Cys Pro Asn Pro Pro Ala IleLeu Asn Gly Arg His 1730 1735 1740 Thr Gly Thr Pro Phe Gly Asp Ile ProTyr Gly Lys Glu Ile Ser Tyr 1745 1750 1755 1760 Ala Cys Asp Thr His ProAsp Arg Gly Met Thr Phe Asn Leu Ile Gly 1765 1770 1775 Glu Ser Ser IleArg Cys Thr Ser Asp Pro Gln Gly Asn Gly Val Trp 1780 1785 1790 Ser SerPro Ala Pro Arg Cys Glu Leu Ser Val Pro Ala Ala Cys Pro 1795 1800 1805His Pro Pro Lys Ile Gln Asn Gly His Tyr Ile Gly Gly His Val Ser 18101815 1820 Leu Tyr Leu Pro Gly Met Thr Ile Ser Tyr Thr Cys Asp Pro GlyTyr 1825 1830 1835 1840 Leu Leu Val Gly Lys Gly Phe Ile Phe Cys Thr AspGln Gly Ile Trp 1845 1850 1855 Ser Gln Leu Asp His Tyr Cys Lys Glu ValAsn Cys Ser Phe Pro Leu 1860 1865 1870 Phe Met Asn Gly Ile Ser Lys GluLeu Glu Met Lys Lys Val Tyr His 1875 1880 1885 Tyr Gly Asp Tyr Val ThrLeu Lys Cys Glu Asp Gly Tyr Thr Leu Glu 1890 1895 1900 Gly Ser Pro TrpSer Gln Cys Gln Ala Asp Asp Arg Trp Asp Pro Pro 1905 1910 1915 1920 LeuAla Lys Cys Thr Ser Arg Ala His Cys Cys Asp Gly Pro Lys Lys 1925 19301935 Lys Lys Lys Lys Ser Pro Ser Lys Ser Ser Gly 1940 1945 53 10 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide53 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10

What is claimed is:
 1. A soluble compound that is directed to an outermembrane of a cell, wherein the soluble compound comprises: (1) asoluble polypeptide that inhibits complement; and (2) a membranelocalization reagent, wherein the membrane localization reagent issoluble and comprises: (a) at least one lipophilic binding elementcomprising aliphatic acyl groups; (b) a hydrophilic peptide bindingelement comprising basic amino acids, wherein the hydrophilic bindingelement is bound to the lipophilic element; and (c) a linker thatcovalently binds a therapeutic agent to the hydrophilic peptide bindingelement of the membrane localization reagent to form the solublecompound.
 2. The soluble compound of claim 1 wherein the hydrophilicpeptide binding element comprises lysine residues.
 3. The solublecompound of claim 1 wherein the hydrophilic peptide binding elementcomprises arginine residues.
 4. The soluble compound of claim 1, whereinthe soluble peptide that inhibits complement is a soluble CD59polypeptide or a soluble DAF polypeptide.
 5. The soluble compound ofclaim 1, wherein the lipophilic binding element and the hydrophilicpeptide binding element each have a dissociation constant of 1 μM to 1mM for a membrane.
 6. The soluble compound of claim 1, wherein thelipophilic binding element and the hydrophilic peptide binding elementeach have a molecular weight of less than 5 kilodaltons.
 7. The solublecompound of claim 1, wherein the soluble compound has a dissociationconstant of 0.01 to 10 nM for a membrane.
 8. A pharmaceuticalcomposition that is directed to an outer membrane of a cell, comprising(1) a soluble polypeptide that inhibits complement; (2) a membranelocalization reagent, wherein the membrane localization reagent issoluble and comprises: (a) at least one lipophilic binding elementcomprising aliphatic acyl groups; (b) a hydrophilic peptide bindingelement comprising basic amino acids, wherein the hydrophilic bindingelement is bound to the lipophilic element; and (c) a linker thatcovalently binds a therapeutic agent to the hydrophilic peptide bindingelement of the membrane localization reagent to form the solublecompound; and (3) a pharmaceutically acceptable carrier or excipient. 9.The pharmaceutical composition of claim 8 wherein the hydrophilicpeptide binding element comprises lysine residues.
 10. Thepharmaceutical composition of claim 8, wherein the hydrophilic peptidebindin element comprises arginine residues.
 11. The pharmaceuticalcomposition of claim 8, wherein the soluble peptide that inhibitscomplement is a soluble CD59 polypeptide or a soluble DAF polypeptide.12. The pharmaceutical composition of claim 8, wherein the lipophilicbinding element and the a hydrophilic peptide binding element each havea dissociation constant of 1 μM to 1 mM for a membrane.
 13. Thepharmaceutical composition of claim 8, wherein the lipophilic bindingelement and the hydrophilic peptide binding element each have amolecular weight of less than 5 kilodaltons.
 14. The pharmaceuticalcomposition of claim 8, wherein the soluble compound has a dissociationconstant of 0.01 to 10 nM for a membrane.